MHC-I restricted epitopes containing non-natural amino acid residues

ABSTRACT

The invention provides for the synthesis of more effective generators of a T-cell immune response by providing peptide derivatives that are MHC class I-restricted antigens. The peptide derivatives of the present invention are prepared or derived from a parent peptide of 8 to 11 amino acid residues in length, wherein the peptide derivative contains a non-natural amino acid substituted in place of a naturally-occurring amino acid residue at one or more primary anchor positions in the parent peptide or at position 6, position 7, or the C-terminus.

1. FIELD OF THE INVENTION

The present invention concerns T-cell antigens and methods of generatinga T-cell immune response against a tumor antigen or a pathogen. Theinvention specifically concerns the synthesis of more effectivegenerators of a T-cell immune response (“T-cell immunogens”).

2. BACKGROUND OF THE INVENTION

The use of peptides as vaccines for the prevention or treatment ofcancer has received considerable attention in recent years as furtherinsight is gained into the steps required to elicit an effective immuneresponse. Even though a greater understanding of these steps has beengained, the success of peptide-based vaccines in the clinic has beenvery limited. This lack of success in treating cancer is probably due tomultiple factors, including the target proteins selected, the nature ofthe peptides chosen from these proteins and the local environment of thetumor. The selection of proteins as targets for the treatment of canceris complicated by the fact that many or most tumor antigens that havebeen identified to date are non-mutated self-antigens, and that theimmune response to these self-antigens is restricted by T-celltolerance. T-cell tolerance can be mediated either centrally orperipherally, and results in the loss of high-avidity cytotoxicT-lymphocytes (CTL) from the immune repertoire that would have thepotential to mediate the killing of tumor cells. Consequently, onechallenge for the development of effective cancer vaccines is toidentify epitopes in tumor antigens to which the immune system has notbeen tolerized or where tolerance can be overcome.

Unlike antibodies that recognize intact soluble or cell-bound proteins,T-cells recognize fragments of proteins (peptides) that are generated byproteolytic degradation. For CTLs, the peptides generally are comprisedof 8 to 11 amino acid residues and are only recognized by cytotoxicT-cells when the peptides are bound to the class I majorhistocompatibility complex (MHCI). MHCIs are expressed by most celltypes and play a critical role in the determination by the immune systemof whether a cell is “self” or “non-self” (Whiteside, T. L. andHerberman, R. B., 1995, Curr. Opin. Immunol. 7, 704-711). The stepsinvolved in processing an antigenic protein to generate peptides thatcan bind to and be presented by MHCIs have been extensively studied.These steps include: (i) internalization of the protein by anantigen-presenting cell (APC); (ii) degradation of the protein intopeptides by the APC proteasome; (iii) translocation of the peptides inthe endoplasmic reticulum by TAP transporters; and (iv) association ofthe peptides with the two chains of an MHCI to form a stableMHCI-peptide complex, which is then exported to the cell surface.

Several factors determine which peptides from an antigenic protein arebound to the MHCI and displayed on the cell surface. The ability to berecognized and cleaved by the proteasome, the stability of the peptidein the cytosol, and the efficiency of transport by the TAP transportersare all critical parameters; however, the most important determinantappears to be the ability of the peptide to bind to the MHCI (Chen etal., 1994, J. Exp. Med. 180, 1471-1483). The topology of the class Ibinding site determines which peptides can bind to the MHCI, and thebinding site topology differs among the different class I molecules.Within the set of peptides able to bind to a given class I molecule,there is typically a range of binding affinities such that those withthe highest affinity compete the best for display at the cell surface.Peptides preferentially selected by these processes for presentation atthe cell surface are referred to as immunodominant epitopes. In general,peptides that bind with high affinity to the MHCI tend to elicitstronger CTL responses than peptides binding with lower affinity (Setteet al., 1994, J. Immunol. 153, 5586-5592). However, it is these higheraffinity, immunodominant epitopes derived from self-proteins (such ascancer antigens) to which T-cells have been exposed and tolerized.

Several investigators have developed approaches for identifying CTLepitopes having low MHCI binding affinity, and increasing the bindingaffinity of these epitopes by making modifications to the native peptidesequence (Parker et al., 1992, J. Immunol. 149, 3580-3587; Tourdot etal., 1997, J. Immunol. 159, 2391-2398; Dionne et al., 2004, CancerImmunol. Immunother. 53, 307-314). These and other investigators haveshown that such modified peptides can be more immunogenic and capable ofeliciting enhanced CTL responses in both in vitro and in vivo assays.

In addition to binding affinity, data from other laboratories have shownthat the stability of the MHCI-peptide complex is important indetermining the immunogenicity of CTL epitopes. Using two CTL epitopesfrom the Epstein-Barr virus nuclear antigen, it was demonstrated thatthe epitope forming the more stable MHCI-peptide complex elicited astronger CTL response when lymphocytes isolated from EBV-seropositivedonors were stimulated with a virus-transformed autologouslymphoblastoid cell line (Levitsky et al., 1996, J. Exp. Med. 183,915-926). From studies examining the binding affinities and dissociationrates of a group of HIV-1 MHCI-binding peptides, it was concluded thatthe immunogenicity of the HIV-1 derived peptides might be predicted moreaccurately by the dissociation rate than by the binding affinity of thepeptide to MHCI (van der Burg et al., 1996, J. Immunol. 156, 3308-3314).Furthermore, in studies examining multiple modified analogs of a murinep53 epitope, it was observed that modifications enhancing the stabilityof the MHCI-peptide complex resulted in significantly more potentimmunogens (Baratin et al., 2002, J. Peptide Sci. 8, 327-334).

Although modification of CTL epitopes can be effective in increasing thebinding affinity of the epitope to the MHCI complex and enhancing theimmunogenicity of the epitope, various investigators have shown thatthese modifications can alter the specificity of the elicited immuneresponse when compared with that of the unmodified “parent” peptide.Substitutions at amino acid “anchor residues” have been shown to alterboth the conformation of the MHCI-peptide complex and the conformationof the contacts with the T-cell receptor (Sharma et al., 2001, J. Biol.Chem. 276, 21443-21449; Denkberg et al. 2002, J. Immunol. 169,4399-4407). Alterations in the sequences of CTL epitopes have also beenshown to change the specificity of the immune response elicited in vitroand in patients treated with the modified epitopes (Clay et al., 1999,J. Immunol. 162, 1749-1755; Yang et al., 2002, J. Immunol. 169, 531-539;Dionne et al., 2003, Cancer Immunol. Immunother. 52, 199-206; Okazaki etal., 2003, J. Immunol. 171, 2548-2555). These results indicate that whenmaking modifications to amino acid sequences of low affinity CTLepitopes, it is critical to assess the impact of these changes on theconformation and flexibility of the modified epitope when bound to theMHCI, as well as on the specificity of the immune response elicited bythe modified epitope.

Starting about a decade ago, techniques were developed that enabled theanalysis of peptides bound to MHCIs (Falk et al., 1991, Nature 351,290-296; Jardetsky et al., 1991, Nature 353, 326-329). Such techniqueshave been used extensively to characterize the peptides that binddifferent MHCI alleles. These studies have led to a general rule thateach class I-bound peptide consists of between 8 and 11 amino acidresidues. Studies have further shown that those peptides capable offorming a complex with a particular MHCI allele have in common thepresence of 2 or 3 largely invariant residues at specific positions inthe peptide. X-ray crystallographic studies have provided structuralinsight into the basis for these conserved residues. These studies haveshown that each MHCI folds to form a groove, and it is within thisgroove that the peptide is displayed. The groove has a specific topologyassociated with each allele, and is characterized by the presence ofseveral depressions or pockets along its length. Crystallographicstudies have shown that the side chains of the conserved peptideresidues (the “anchor residues”) extend into these pockets, and that theinteraction between the MHCI and peptide within these pockets supplies asignificant portion of the energy of binding. As such, these largelyinvariant, allele-specific residues are referred to as “primary anchorresidues.” Interactions between side chains of amino acids near thetermini of the peptide are also important for binding of the peptide tothe MHCI, and they are observed in the crystallographic studies to buryinto the ends of the MHCI binding groove. Much of the range in bindingaffinities among peptides that bind to a given MHCI allele is due tosequence variations in this array of primary anchor residues. However,it is also clear that other residues outside the primary anchorpositions can contribute to MHCI binding (Kondo et al., 1995, J.Immunol. 155, 4307-4312; Schonbach et al., 1995, J. Immunol. 154,5951-5958), and these other residues are referred to as “secondaryanchor residues”. Secondary anchor residues may also contribute to therange in binding affinities among peptides binding to a particular MHCI.

A number of investigators have successfully enhanced the immunogenicityof both viral and tumor peptide epitopes by increasing the affinity ofthe peptide for the MHCI. Typically, this has been achieved by replacingsub-optimal residues found at the primary anchor positions with anoptimal natural residue. In mice, an anchor-modified epitope from mutantRas binds more effectively to the H-2K^(d) allele, induces enhancedcytolytic activity in vitro, and elicits a greater T-cell response invivo than the unmodified parent mutant Ras peptide (Bristol et al.,1998, J. Immunol. 160, 2433-2441). Similar results were also observed inmice transgenic for human HLA-A2. In these experiments, ananchor-modified epitope from HIV reverse transcriptase was shown to bemore effective at inducing CTL reactive with the reverse transcriptasethan the parent peptide, and afforded greater protection in vivo againsta challenge with Vaccinia virus expressing the HIV-1 reversetranscriptase (Okazaki et al., 2003, J. Immunol. 171, 2548-2555). Inaddition, anchor-modified epitopes from the melanoma antigen gp100 wereshown to bind with higher affinity to HLA-A*0201 than did the parentepitopes, and induced melanoma-reactive CTL in peripheral bloodleukocytes (PBL) isolated from 7 of 7 HLA-A2⁺ melanoma patients. Incontrast, the unmodified parent epitopes induced melanoma-reactive CTLin PBL from only 2 of the 7 patients (Parkhurst et al., 1996, J.Immunol. 157, 2539-2548).

Alternative approaches to enhancing the binding affinity of CTL epitopesto MHCI include amino acid substitutions at positions other than theprimary anchor residues. Systematic analyses of the role of secondaryanchor residues in the binding of CTL epitopes to the MHCI have beenperformed, revealing that amino acid residues at positions other thanthe primary anchor residues can strongly influence the binding of theepitope (Deres et al., 1993, Cell. Immunol. 151, 158-167; Kondo et al.,1995, J. Immunol. 155, 4307-4312). In addition, studies examining theimmunogenicity of two epitopes from ovalbumin indicate that amino acidresidues at sites other than the primary anchor sites can significantlyinfluence binding and presentation of an epitope (Chen et al., 1994, J.Exp. Med. 180, 1471-1483). A more general strategy has also beenproposed to enhance the binding and immunogenicity of epitopes that bindwith low affinity to HLA-A2.1. Studies with multiple low-affinityHLA-A2.1 epitopes show that the introduction of tyrosine at the firstposition can enhance the binding affinity and immunogenicity of themodified epitope (Pogue et al., 1995, Proc. Natl. Acad. Sci. USA 92,8166-8170; Toudot et al., 2000, Eur. J. Immunol. 30, 3411-3421; PatentApplication Publication US 2004/0072240 A1 by Kosmatopoulos et al.).

Non-natural amino acids have also been used with some success to replacenatural amino acids and increase the immunogenicity of CTL epitopes.Using an antigenic peptide from the influenza virus matrix protein, aretro-inverso amide bond substitution was shown to enhance binding ofthe modified peptide to HLA-A2; however, this retro-inverso modificationwas only effective when inserted between Position 1 (P1) and Position 2(P2) (as numbered from the amino terminus of the peptide), and resultedin a significant reduction in binding when used between P2 and P3, or P3and P4, or P5 and P6, or P8 and P9 (Guichard et al., 1996, J. Med. Chem.39, 2030-2039).

An alternative approach utilized an epitope from Melan-A (GenBank No.U06654). Melan-A is unrelated to any known gene and is encoded by anapproximately 18-kilobase gene comprised of 5 exons (Coulie et al.,1994, J. Exp. Med. 180, 35-42). Similar to other melanoma-associatedantigens, Melan-A is expressed in most melanoma tumor samples and normalmelanocytes. HLA-A2 restricted melanoma-specific CTLs from nine of tenpatients recognize primarily the MART-1 epitope, i.e., amino acidresidues 27-35 (AAGIGILTV; SEQ ID NO:1) of Melan-A. Kawakami, Y., etal., J. Exp. Med. 180, 347-352 (1994). The MART-1 epitope of Melan-A hasbeen studied extensively and has been used as a model system forinvestigating cellular immune responses in humans. Valmori et al., J.Immunol. 160, 1750-1758 (1998). The alternative approach involved thesubstitution of a β-amino acid at Position 4 (P4), resulting in enhancedbinding of the peptide to HLA-A2 and increased stability of thepeptide-MHCI complex. However, only one out of three MART-1 specific CTLclones isolated from HLA-A2 restricted human tumor infiltratinglymphocytes (TIL) recognized and lysed target cells pulsed with themodified peptide. Guichard et al., J. Med. Chem. 43:3803-3808 (2000).

In an attempt to reduce biodegradation and enhance the immunogenicity ofCTL epitopes, 36 peptide derivatives of the MART-1 epitope were designedbased on knowledge of the degradation pathway in human serum.Non-natural amino acids were substituted at positions 1, 2, 8, 9 and 10of the 10 amino acid MART-1 epitope individually and in combination andthese modified MART-1 epitopes were then tested for their resistance toproteolysis and antigenic properties (Blanchet et al., 2001, J. Immunol.167, 5852-5861). Eight of the modified epitopes were found to haveenhanced stability against degradation when incubated in human serum,and three of these analogs were shown to be more potent than theparental epitope in stimulating in vitro MART-1 specific CTL responsesin PBMC from normal donors. Non-natural amino acids have also been usedin studies aimed at enhancing the immunogenicity of a poorly immunogenicepitope from murine p53. By replacing cysteine with aminobutyric acid atpositions 4 and 8 and methionine with norleucine at positions 3 and 9 inthe epitope, the modified peptides bound with higher affinity to MHCIand appeared to be more potent immunogens. Baratin et al., J. PeptideSci. 8:327-334 (2002).

With a strong scientific rationale, highly promising preclinical resultsand the frequently observed induction of target-specific immuneresponses in treated patients, the interest in and efforts directed atthe development of effective cancer vaccines continues to increase. Arecent study described 645 clinical trials related to cancer vaccinesand reported that new cancer vaccine trials had shown a steady increasesince 2001 reaching more than 60 new trials each year (Cao, X., et al.,2008, Immunome Research 4, 1-11). However, the results from theseclinical trials have been much less encouraging with only rareoccurrences of objective tumor regression despite the detection in somepatients of robust target-specific immune responses (Mocellin, S., etal., 2009, Curr. Med. Chem. 16, 4779-4796). Investigators haveidentified a number of factors that likely contribute to the poorclinical results including the selection of suboptimal targets, thepresence of mutations in tumor cells that can promote tumor escape andimmunosuppressive factors including cells, proteins and chemicalspresent in the tumor environment. It has been proposed that a successfulcancer vaccine will need to address these and other factors byincorporating at least one and most likely multiple optimized epitopes,enhanced delivery systems, adjuvants and co-factors to activatecytotoxic and helper T-cells as well as antigen presenting cells, andstrategies to inhibit the immunosuppressive network (Kanodia, S. andKast, W. M., 2008, Expert Rev. Vaccines 7, 1533-1545).

There is a continuing need in the art for improved methods of generatinga T-cell immune response.

3. SUMMARY OF THE INVENTION

The present invention provides a peptide derivative that is an MHC classI (“MHCI”) restricted antigen, and which is prepared or derived from aparent peptide of 8 to 11 amino acid residues in length, and preferablyeither 9 or 10 amino acid residues in length, wherein the peptidederivative contains a non-natural amino acid substituted in place of anaturally occurring amino acid residue at one or more primary anchorpositions, for example at one primary anchor position, or at two primaryanchor positions. Thus, the invention provides a peptide derivative of aMHCI restricted parent antigen (e.g., parent peptide or parent epitope)comprising a non-natural amino acid at at least one anchor position.

The invention further provides a peptide derivative of a MHCI restrictedparent antigen comprising a non-natural amino acid at one anchorposition and a second substitution at a second position. The secondsubstitution at a second position may be at a second anchor position, ormay be at the C-terminus (PΩ), e.g., for MHCI peptides that do not havean anchor position at the C-terminus, or may be at position 6 (P6) orposition 7 (P7).

Any of the peptide derivatives provided by the present invention may ormay not be in the form of a pharmaceutically acceptable salt.

The parent peptide is an MHCI restricted antigen and the peptidederivative provided by the present invention is a MHCI restrictedantigen that binds at least the same MHCI molecule as the parentpeptide, e.g., if the parent peptide binds HLA-A*0201, then the peptidederivative also binds HLA-A*0201. In addition, the peptide derivative ofthe present invention is able to trigger an expansion of T-cells thatare able to bind the parent peptide when it is complexed with MHCI.

The peptide derivatives of the present invention may also have increasedimmunogenicity in comparison to the parent peptide. In preferredembodiments, the peptide derivative exhibits at least one, or at leasttwo, or at least three, or at least four, or all five of the followingproperties.

A first property is that the peptide derivative of the present inventiongenerates a T-cell immune response that is greater than the T-cellimmune response generated by the parent peptide. In one embodiment, theparent peptide generates a detectable T-cell immune response, but thepeptide derivative generates a T-cell immune response which is greaterthan the T-cell immune response generated by the parent peptide. Inanother embodiment, the parent peptide does not generate a detectableT-cell immune response, whereas the peptide derivative of the presentinvention generates a T-cell immune response that can be detected. Inadditional embodiments, the immune response may be T-cell lysis oftarget cells, cytokine release, and/or T-cell degranulation.

A second property is that the peptide derivative of the presentinvention binds to MHCI with an affinity that is higher than theaffinity with which the parent peptide binds to MHCI, i.e., the peptidederivative has a lower K_(D) than the parent peptide.

A third property is that the affinity of T-cell receptors for thecomplex formed between MHCI and a peptide derivative of the presentinvention is higher than the affinity of T-cell receptors for thecomplex formed between MHCI and the parent peptide. This increasedaffinity may be determined using a tetramer assay (Laugel, B., et al.,2007, J. Biol. Chem. 282, 23799-23810; Holmberg, K., et al., 2003, J.Immunol. 171, 2427-2434; Yee, C., et al., 1999, J. Immunol. 162,2227-2234).

A fourth property is that a complex formed between MHCI and a peptidederivative of the present invention is more stable (i.e., has a sloweroff-rate) than a complex formed between MHCI and the parent peptide.

A fifth property is that the peptide derivative of the present inventiontriggers an expansion of a broader number of T-cell clones thatrecognize the parent peptide than are triggered by the parent peptide.

In a preferred embodiment, the parent peptide is from 8 to 11 amino acidresidues in length, and preferably either 9 or 10 amino acid residues inlength.

In one embodiment, the parent peptide is a nonamer (i.e., consisting of9 amino acid residues).

In another embodiment, the parent peptide is a decamer (i.e., consistingof 10 amino acid residues).

In another embodiment, the substituted amino acid residue is at the P2anchor position in the parent peptide.

In another embodiment, the parent peptide is a nonamer wherein thesubstituted amino acid residue is at the P2 anchor position, or adecamer wherein the substituted amino acid residue is at the P2 anchorposition.

In one embodiment, the peptide derivative of the present invention issubstantially purified, i.e., comprised in a preparation in which thepeptide derivative is at least about 70% by weight of the totalpreparation.

A peptide derivative of the present invention can be further modified soas to enhance one or more beneficial properties thereof, including anyone or more of the aforementioned five properties listed above, oranother property such as solubility or in vivo half-life, among others.In one non-limiting example, a peptide derivative of the presentinvention is conjugated to a polyethylene glycol (PEG) molecule ofappropriate molecular weight so as to increase the in vivo half-life ofthe peptide derivative.

The present invention further provides a pharmaceutical compositioncomprising any of the aforementioned peptide derivatives combined with apharmaceutically acceptable carrier. The pharmaceutical composition maybe adapted for administration by any appropriate route, including byparenteral administration.

The present invention further provides a method of preparing apharmaceutical composition comprising admixing any of the aforementionedpeptide derivatives with a pharmaceutically acceptable carrier.

The present invention further provides a complex comprising an MHCIhaving a peptide derivative of the present invention bound within itsantigen-binding groove.

The present invention further provides a cell comprising an immunogeniccell-surface bound complex consisting of MHCI having a peptidederivative of the present invention bound within its antigen-bindinggroove. The cell can be any cell expressing MHCI, either naturally or asthe result of genetic engineering.

The present invention further provides a method for identifying apeptide derivative that has improved ability to activate and expand aclone of a T-cell, which method comprises:

-   -   (a) preparing a peptide derivative of a parent peptide, wherein        the parent peptide is 8 to 11 amino acid residues in length, and        preferably 9 or 10 amino acid residues in length, such that the        peptide derivative has a non-natural amino acid substituting for        an amino acid residue at one or more primary anchor positions in        the parent peptide; and    -   (b) determining whether the peptide derivative of step (a) is        more effective than the parent peptide at activating and        expanding one or more T-cell clones.

In one embodiment, the parent peptide can detectably activate and expandthe clone of the T-cell, but not as effectively as the peptidederivative.

In another embodiment, the parent peptide cannot detectably activate andexpand the clone of the T-cell, whereas the peptide derivative candetectably activate and expand the clone of the T-cell.

The present invention further provides a method for identifying apeptide derivative that has an affinity to MHCI that is higher than theaffinity of its parent peptide to MHCI, which method comprises:

-   -   (a) preparing a peptide derivative of a parent peptide, wherein        the parent peptide is 8 to 11 amino acid residues in length, and        preferably 9 or 10 amino acid residues in length, such that the        peptide derivative has a non-natural amino acid substituting for        an amino acid residue at one or more primary anchor positions in        the parent peptide; and    -   (b) determining whether the peptide derivative of step (a) has        an affinity for MHCI that is higher than the affinity of the        parent peptide for MHCI.

The present invention further provides a method for identifying apeptide derivative, wherein there is a higher affinity of T-cellreceptors for the complex formed between MHCI and the peptide derivativethan for the complex formed between MHCI and the parent peptide of thepeptide derivative, which method comprises:

-   -   (a) preparing a peptide derivative of a parent peptide, wherein        the parent peptide is 8 to 11 amino acid residues in length, and        preferably 9 or 10 amino acid residues in length, such that the        peptide derivative has a non-natural amino acid substituting for        an amino acid residue at one or more primary anchor positions in        the parent peptide; and    -   (b) determining whether there is a higher affinity of T-cell        receptors for the complex formed between MHCI and the peptide        derivative than for the complex formed between MHCI and the        parent peptide.

The present invention further provides a method for identifying apeptide derivative that forms a complex with MHCI that is more stablethan the complex formed between the parent peptide and MHCI, whichmethod comprises:

-   -   (a) preparing a peptide derivative of a parent peptide, wherein        the parent peptide is 8 to 11 amino acid residues in length, and        preferably 9 or 10 amino acid residues in length, such that the        peptide derivative has a non-natural amino acid substituting for        an amino acid residue at one or more primary anchor positions in        the parent peptide; and    -   (b) determining whether the peptide derivative of step (a) forms        a complex with MHCI that is more stable than the complex formed        between the parent peptide and MHCI.

The present invention further provides a method for identifying apeptide derivative that can trigger an expansion of T-cells able torecognize the parent peptide of the peptide derivative, which methodcomprises:

-   -   (a) preparing a peptide derivative of a parent peptide, wherein        the parent peptide is 8 to 11 amino acid residues in length, and        preferably 9 or 10 amino acid residues in length, such that the        peptide derivative has a non-natural amino acid substituting for        an amino acid residue at one or more primary anchor positions in        the parent peptide; and    -   (b) determining whether the peptide derivative of step (a) can        trigger an expansion of T-cells that are able to recognize the        parent peptide of the peptide derivative.

The present invention further provides a method for identifying apeptide derivative that can trigger an expansion of a broader number ofT-cell clones that recognize the parent peptide than can be triggered bythe parent peptide, which method comprises:

-   -   (a) preparing a peptide derivative of a parent peptide, wherein        the parent peptide is 8 to 11 amino acid residues in length, and        preferably 9 or 10 amino acid residues in length, such that the        peptide derivative has a non-natural amino acid substituting for        an amino acid residue at one or more primary anchor positions in        the parent peptide; and    -   (b) determining whether the peptide derivative of step (a) can        trigger an expansion of a broader number of T-cell clones that        recognize the parent peptide than can be triggered by the parent        peptide.

A peptide identified by any of the aforementioned identification methodswill exhibit at least one, and preferably at least two, or at leastthree, or at least four, or all five of the improved properties recitedin the aforementioned identification methods.

In one embodiment of any of the aforementioned identification methods,the parent peptide is a nonamer.

In another embodiment of any of the aforementioned identificationmethods, the parent peptide is a decamer.

In another embodiment of any of the aforementioned identificationmethods, the substituted amino acid residue is at the P2 anchorposition.

In another embodiment of any of the aforementioned identificationmethods, the parent peptide is a nonamer wherein the substituted aminoacid residue is at the P2 anchor position, or a decamer wherein thesubstituted amino acid residue is at the P2 anchor position.

The present invention further provides a method of using a peptidederivative of the present invention to activate and expand a T-cellclone that is reactive towards a parent peptide. The activated andexpanded T-cells can be used to recognize the parent peptide in asubject. This method of use can be practiced by administering thepeptide derivative directly to the subject. Alternatively,dendritic-type antigen presenting cells (“dAPCs” or “DCs”) can becultured ex vivo and pulsed with the peptide derivative, and the pulseddAPCs administered directly to the subject to activate and expandT-cells in vivo. Alternatively, a subject's T-cells can be cultured exvivo, such that activated expanded T-cells can be administered directlyback to the subject. Such methods can be used to obtain a therapeuticbenefit in the subject so as to, e.g., treat or prevent a condition suchas a cancer or infection. The activated and expanded T-cells can also beused to recognize the parent peptide in a biological sample ex vivo.

The present invention further provides a method of inducing an immuneresponse against a peptide derivative of the present invention. Thepresent invention also provides a method of inducing an immune responseagainst a parent peptide using a peptide derivative of the presentinvention. The peptide derivative may be administered to a subject toinduce an immune response, or may be contacted with immune cells from asubject ex vivo. The peptide derivative may be part of a pharmaceuticalor diagnostic composition.

The present invention further provides a method for treating orpreventing a condition in a subject, comprising administering atherapeutically effective amount of a peptide derivative of the presentinvention to a subject in need of such treatment. In one embodiment,said method is for treating said condition in a subject. In oneembodiment, the peptide derivative administered to the subject ispresent in a pharmaceutical composition provided by the presentinvention. The present invention also provides the peptide derivative ofthe present invention for use as a medicament.

The condition treated or prevented by a method or medicament of thepresent invention may be selected from the group consisting of cancersand infections as described further herein. In one embodiment, thecancer is treated by a method of the present invention. In anotherembodiment, the cancer is prevented by a method of the presentinvention. In one embodiment, the infection is treated by a method ofthe present invention. In another embodiment, the infection is preventedby a method of the present invention.

The present invention further provides a use of any of the peptidederivatives of the present invention in the manufacture of a medicamentto treat a condition, such as a cancer or an infection, in a subject.

The present invention further provides the peptide derivative of thepresent invention for use as a medicament, and specifically for use inthe treatment or prevention of a cancer or of an infection, and inparticular for use in the treatment of a cancer or of an infection.

4. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a peptide derivative that is an MHCIrestricted antigen, and is derived from a parent peptide 8 to 11 aminoacid residues in length, preferably 9 or 10 amino acid residues inlength, wherein the peptide derivative contains a non-natural amino acidsubstituted in place of a naturally occurring amino acid residue at oneor more primary anchor positions, preferably at two primary anchorpositions, and more preferably at one primary anchor position. Thus, theinvention provides a peptide derivative of a MHCI restricted parentantigen (e.g., parent peptide or parent epitope), the derivativecomprising a non-natural amino acid in at least one primary anchorposition. In some embodiments, the anchor position is Position 2 (P2).In some embodiments, the anchor position is at the C-terminus (PΩ). Insome embodiments, there is a non-natural amino acid both in position P2and in position PΩ. See below for additional anchor positions which canbe taken by a non-natural amino acid in some embodiments.

The invention further provides a peptide derivative of a MHCI restrictedparent antigen comprising a non-natural amino acid at one anchorposition, e.g. at position P2, and a second substitution at a secondposition. In some embodiments, the second position is position 6 (P6) orposition 7 (P7). In some embodiments, the second position is theC-terminus (PΩ), e.g., for MHCI peptides that do not have an anchorposition at the C-terminus.

The peptide derivative provided by the present invention is a MHCIrestricted antigen that binds at least the same MHC molecule as theparent peptide, e.g., if the parent peptide binds HLA-A*0201, then thepeptide derivative also binds HLA-A*0201. In addition, the peptidederivative of the present invention is able to trigger an expansion ofT-cells that are able to bind the parent peptide when it is complexedwith MHCI. A peptide derivative of the invention may also have improvedimmunogenicity over its parent peptide. The peptide derivatives of theinvention do not include the peptides disclosed in the followingpublications, each of which is incorporated herein by reference in itsentirety: Bianco, J. Biol. Chem. 273:28759-65 (1998); Bolin, J. Med.Chem. 43:2135-48 (2000); Dedier, J. Receptor & Signal Transduct. Res. 19(1-4):645-57 (1999); Douat-Casassus, J. Med. Chem. 50:1598-1609 (2007);Gomez-Nunez, PLoS ONE 3(12):e3938 (2008); Haro, Kurtis J.,“Photo-Reactive and Non-Natural Amino Acid Epitopes of Human WT1 EnhanceImmunogenicity and Allow Kinetic Study of Antigen Processing,” ASHAnnual Meeting Abstracts, Part 1, Vol. 118(11):684a, abst. 2311 (2007);Jones, J. Pept. Sci. 14:313-20 (2008); Liu, US 2006/0057673 A1 (2006);Poenaru, J. Med. Chem. 42:2318-31 (1999); Rovero, J. Pept. Sci. 1:266-73(1995); Schultz, U.S. Pat. No. 6,716,809 B1 (2004); Baratin, J. Pept.Sci. 8:327-34 (2002); Blanchet et al., J. Immunol. 167:5852-61 (2001);Guichard et al., J. Biol. Chem. 270:26057-59 (1995); Guichard et al., J.Med. Chem. 39:2030-39 (1996); Guichard et al., J. Med. Chem. 43:3803-8(2000); Herve et al., Mol. Immunol. 34:157-63 (1997); Kanodia and Kast,Expert Rev. Vaccines 7:1533-45 (2008); Krebs and Rognan, Pharmaceut.Acta Helv. 73:173-81 (1998); Krebs et al., J. Peptide Sci. 4:378-88(1998); Krebs et al., J. Biol. Chem. 273:19072-9 (1998); Marschutz etal., Peptides 23:1727-33 (2002); MacGregor, Curr. Opin. Pharmacol.8:616-19 (2008); Meziere et al., J. Immunol. 159:3230-37 (1997);Mocellin, et al., Curr. Med. Chemistry 16:4779-96 (2009); Peonaru etal., J. Med. Chem. 42:2318-31 (1999); Purcell et al., Nat. Rev. DrugDiscov. 6:404-14 (2007); Rhagavan et al., Protein Sci. 5:2080-88 (1996);Rognan et al., Biochemistry 33:11476-85 (1994); Rognan et al, PNAS92:753-57 (1995); Rognan et al., J. Computer-Aided Mol. Design 11:463-78(1997); Rognan, “Molecular modelling of protein-peptidecomplexes—Application to major histocompatibility proteins,”Habilitationsschrift, Eidgenossiche Technische Hochsschule (ETH) Zurich(1998); Rognan et al., J. Med. Chem. 42:4650-58 (1999); Rovero et al.,PNAS 92:753-57 (1995); Scapozza et al., Acta Crystallogr. D Biol.Crystallogr. 51(Pt 4):541-9 (1995); Steer et al., Curr. Med. Chem.9:811-22 (2002); von Grafenstein, Pharm. Res. 13(9)(Suppl):S-139, abstr.MNPC 5002 (1996); Webb et al., J. Immunology 175:3810-18 (2005); Weisset al., Chem. and Biol. 2:401-7 (1995); Weiss et al., PNAS 93:10945-48(1996); WO 03/084467 (Euro-Celtique); Rhuby et al., Biochem. J.268:249-62 (1990); Kazmierski et al., J. Am. Chem. Soc. 113:2275-83(1991); Hohsaka et al., Curr. Opinion Chem Biol 6:809-15 (2002); Kahn,U.S. Pat. No. 5,440,013; Nicolette, U.S. Pat. No. 6,338,945 and U.S.Pat. No. 6,579,970; and WO 2006/138562 (Mannkind Corporation).

4.1. Definitions

As used herein, the term “naturally occurring amino acid residue” or“natural amino acid” refers to one of the L-amino acids occurringnaturally in proteins. As used herein, the term “natural amino acid sidechain” refers to a side chain attached to the Cα of one of the naturalamino acids. Unless otherwise indicated, the “naturally-occurring aminoacid residues” in the peptide sequences disclosed herein are presentedusing the standard single letter amino acid abbreviations for the twentynaturally occurring amino acids, as follows:

TABLE 1 Amino Acid Abbreviation Alanine A Arginine R Asparagine NAspartic Acid D Cysteine C Glutamic Acid E Glutamine Q Glycine GHistidine H Isoleucine I Leucine L Lysine K Methionine M Phenylalanine FProline P Serine S Threonine T Tryptophan W Tyrosine Y Valine V

As used herein, the term “non-natural amino acid” refers to amino acidsthat do not naturally occur in proteins, and that contain, by analogy tonaturally occurring amino acids, a primary amine, a free carboxylategroup and a side chain and that can be inserted into a peptide sequenceusing conventional peptide synthesis methods. In some embodiments, anon-natural amino acid is conformationally constrained. In someembodiments, a non-natural amino acid has a side chain that is the sameas or chemically similar to the side chain of the residue that itreplaces.

In some embodiments, a “non-natural amino acid” contains a non-naturalside chain. In some embodiments, a non-natural side chain isconformationally constrained. In some embodiments, a non-natural sidechain is similar to the natural side chain of the residue that itreplaces.

A used herein, the terms “conformationally constrained,” “conformationconstraining,” “conformational constraint,” and the like, as usedherein, when referring to a non-natural amino acid, generally refer to anon-natural amino acid used to replace (i.e., substitute for) an aminoacid residue in a parent peptide such that the peptide resulting fromsuch substitution has less flexibility (i.e., is more conformationallyconstrained than the parent peptide), as evidenced by increasedconstraint in the rotation about one or more polymer bond, e.g., bydisubstitutions of C^(α) or a substitution at the amide nitrogen, or byfewer rotational degrees of freedom of all available peptide bonds inthe backbone, of the peptide derivative. For example, a non-naturalamino acid residue provides a conformational constraint relative to theoriginal residue at that position when the rotations about backbonedihedral angles (φ,ψ) are constrained by substitutions at C^(α), e.g.,αMe-leucine, or at the amide nitrogen.

In some embodiments, the “conformationally constrained” non-naturalamino acid is one that, upon substitution into an anchor position, e.g.,the P2 position, of the parent peptide, results in the adoption ofPhi/Psi backbone angles of −60±30 degrees, and +110±50 degrees,respectively. The adoption of these backbone angles can be determined bystandard techniques, including by NMR, X-ray crystallography, orcomputational structural prediction.

The number of rotational degrees of freedom of all available bonds inthe backbone is determined by the number of non-cyclic single bondsconnecting multivalent atoms having at least two distinct substituentsor having aromatic bonds. For example, a proline residue has one degreeof freedom; glycine and alanine have two; serine and valine have three;phenylalanine, leucine and aspartic acid have four; methionine andglutamine have five; and lysine and arginine have six.

The following non-natural amino acids have side chains that are similarto leucine (e.g., the most preferred naturally-occurring amino acid as aP2 anchor in HLA-A0201) and would be suitable for replacing leucine in aparent peptide (e.g., for substitution of a P2 leucine in a HLA-A0201restricted peptide epitope). They are grouped by the number of degreesof freedom of all available bonds in the backbone: two degrees,aminocyclopropylcarboxylic acid (“ACC”), aminocyclobutylcarboxylic acid(“ACBC”), aminocyclopentylcarboxylic acid (“ACPC”), andaminocyclohexylcarboxylic acid (“ACHC”); three degrees,L-cyclohexylglycine, L-cyclopentylglycine, and L-phenylglycine; fourdegrees, β-cyclopropylalanine, β-amino-L-n-butyric acid (homoalanine),L-4,5-dehydroleucine, and L-norvaline; and five degrees,L-styrylalanine, and L-norleucine.

Of the foregoing, the amino acids with three or fewer degrees of freedomare conformationally constrained compared, e.g., to leucine andmethionine, while those with four degrees of freedom areconformationally constrained compared only to methionine.

As a further example, the replacement of glutamic acid by1,3-dicarboxyl, 1-amino-cyclobutane would be an example of asubstitution of a conformationally constrained residue having ananalogous side chain.

As used herein, a “conformationally constrained side chain” is any sidechain with fewer degrees of freedom than the side chain of the residuethat it replaces.

As used herein, the term “conformationally constrained peptide” means apeptide that results from the replacement of an amino acid in a parentpeptide with a more conformationally constrained non-natural amino acid.

In one embodiment, the non-natural amino acid is a Cα disubstitutedamino acid. The Cα and the substituents may or may not form a ring. Inanother embodiment, the non-natural amino acid is an N-substituted aminoacid. The non-natural amino acid may be selected from the groupconsisting of chg, cpg, ACC, ACBC, ACPC, ACHC, phg, β-cp-Ala, styr-Ala,Nle, Abu, γ,δ-Δ-Leu, Nrv, c3a, c5g, dfb and dhl. In one embodiment, thisis under the proviso that the peptide is not ENrvAGIGILTV orELAGIGILTNrv. In another embodiment, the peptide is not a peptideselected from the group consisting of ENrvAGIGILTV, ELAGIGILTNrv,ENrvAGIGILTNrv, ENrvAGIGILTNle, ENleAGIGILTV, ENleAGIGILTNrv,ENleAGIGILTNle, ELAGIGILTNle, EAAGIGILTNrv, and EAAGIGILTNle. In anotherembodiment, the non-natural amino acid may be selected from the groupconsisting of chg, cpg, ACC, ACBC, ACPC, ACHC, phg, β-cp-Ala, styr-Ala,Abu, γ,δ-Δ-Leu, c3a, c5g, dfb and dhl.

In another embodiment, the non-natural amino acid is selected from thegroup consisting of chg (L-cyclohexylglycine), cpg(L-cyclopentylglycine), ACC, ACBC, ACPC, ACHC, phg (L-phenylglycine),β-cyclo-propylalanine (β-cp-Ala), styrylalanine (styr-Ala), norleucine(Nle), β-amino-L-n-butyric acid (Abu), γ,δ-dehydroleucine (γ,δ-Δ-Leu),and norvaline (Nrv). In one embodiment, this is under the proviso thatthe peptide is not ENrvAGIGILTV or ELAGIGILTNrv. In another embodiment,the peptide is not a peptide selected from the group consisting ofENrvAGIGILTV, ELAGIGILTNrv, ENrvAGIGILTNrv, ENrvAGIGILTNle,ENleAGIGILTV, ENleAGIGILTNrv, ENleAGIGILTNle, ELAGIGILTNle,EAAGIGILTNrv, and EAAGIGILTNle.

In another embodiment, the non-natural amino acid is selected from thegroup consisting of c3a [beta-cyclopropylalanine], c5g [aminocyclopentylcarboxylic acid], chg [L-cyclohexylglycine], cpg [L-cyclopentylglycine],dfb [L-6,6-difluoro-bicyclo[3.1.0]hexylglycine], dhl[L-isopropenealanine], phg [L-phenylglycine], and sta [L-styrylalanine].

As used herein, the “[c3a]” refers to the following chemical structures:

As used herein, the “[c5g]” refers to the following chemical structures:

As used herein, the “[chg]” refers to the following chemical structures:

As used herein, the “[cpg]” refers to the following chemical structures:

As used herein, the “[dfb]” refers to the following chemical structures:

As used herein, the “[dhl]” refers to the following chemical structures:

As used herein, the “[phg]” refers to the following chemical structures:

As used herein, the “[sta]” refers to the following chemical structures:

As used herein, the term “MHCI” means “Class I Major HistocompatibilityComplex”, and can be selected from those listed in Tables 6 and 7,below. As used herein, the term “HLA” refers to Human Leukocyte Antigenand is the name of the major histocompatibility complex (MHC) in humans.HLA is important in immune functions including the differentiationbetween self and non-self cells and antigen presentation.

As used herein to refer to a peptide, the term “wild type” refers to theamino acid sequence of a naturally occurring peptide. As used herein torefer to an amino acid residue, the term “wild type” refers to the aminoacid residue that occurs naturally at a particular position in anaturally occurring peptide.

The “parent peptide” (also referred to herein as “parent epitope” and“MHC restricted antigen”) is a putative MHCI epitope. Parent peptidescan be identified using computer programs that make predictions based onthe presence or absence of the conserved residues at both primary andsecondary anchor sites. There are programs that consider all of thepositions in a peptide to predict whether it is a MHCI peptide. Parentpeptides can also be identified experimentally using peptides preparedsynthetically and then tested for their ability to bind to the MHCIprotein complex. Parent peptides may also be identified by extractingpeptides from the MHC I molecules of a population of cells. See belowfor methods to identify parent peptides. It has also been verifiedexperimentally that many naturally occurring MHCI epitopes do not havethe consensus anchor residues.

The “parent peptide” can be any peptide, but preferably is a peptideobtained from, or otherwise identified as part of, synthesized by, orassociated with, a target antigen of a cancer cell or infectious agentor a cell infected with an infectious agent (e.g., a peptide encoded byan infectious agent or a self-peptide whose expression is induced by theinfectious agent). The parent peptide may be a peptide that occursnaturally, either by itself or as a portion of a larger proteinmolecule, i.e., a target antigen. A parent peptide may be a naturallyoccurring peptide (either by itself or as a portion of a targetantigen), i.e., a “wild type” peptide, or a parent peptide may be ananalog of a wild type peptide. The expression of the parent peptide (orthe larger target antigen of which it may be part) may be unique to acancer cell or to a particular infectious agent. Alternatively, theparent peptide (or the target antigen of which it may be part) may beexpressed by both cancer and non-cancer cells in a subject, but may berelatively over-expressed by the cancer cells, or may be common to boththe infectious agent and the subject, but may be relativelyover-expressed by the infectious agent. Alternatively, the parentpeptide may not be naturally occurring, but may be produced through oneor more synthetic schemes. In either case, the term “parent peptide”will generally refer to the immediate precursor of a peptide derivativeof the present invention prior to substitution with a non-natural aminoacid, e.g., a conformation-constraining non-natural amino acid residue,according to the present invention.

The “parent peptide” and the “peptide derivative” of the presentinvention are identical except for the substitution of one or morenon-natural amino acids as described above (e.g., at P2 and/or at P6 orP7 or PΩ for HLA-A2). The parent peptide may or may not have the aminoacid sequence of a wild type (i.e., naturally occurring) peptide. Forexample, a peptide having a wild type amino acid sequence may bedirectly modified according to the present invention to replace thenative amino acid residue at the P2 position with a non-natural aminoacid, in which case the wild type peptide is the “parent peptide” of thepeptide derivative. Alternatively, a peptide having a non-wild typeamino acid sequence (referred to herein as an analog) may be modifiedaccording to the present invention to substitute a non-natural aminoacid for the amino acid residue at, e.g., the P2 position, in which casea non-wild type peptide is the “parent peptide” of the peptidederivative. An example of a non-natural peptide (analog), which may be aparent peptide in the present methods, is a heteroclitic analog, e.g.,as described in US 20060018915 A1.

The term “anchor position” refers to certain amino acid positions withina peptide that bind to a MHCI molecule where one or a small number ofamino acid residues are found almost exclusively, i.e., they areconserved or semi-conserved across a population of epitopes. The aminoacids in these positions are said to “anchor” the peptide into thebinding groove of the MHCI molecule by having side chains that arecomplementary to pockets present in the binding groove. One of thesepockets usually binds the carboxy-terminal amino acid of a 8, 9, 10, or11 amino acid long peptide. The other positions of the anchor residuesare different between the MHCI alleles, but frequently are the second,third or fifth residue from the amino terminus (see, e.g., Tables 6 and7, below). For peptides that bind to HLA-A2.1, the anchor residues areat positions P2 (i.e., position 2 numbering sequentially from the aminoterminal amino acid) and PΩ (the carboxy-terminal position that caneither be P9 (position 9) or P10 (position 10), depending on the lengthof the peptide).

Peptide derivatives can be designed according to the methods of theinvention from a parent peptide, without regard to the MHC binding motifor supermotif to which the parent peptide belongs. The primary anchorresidues of the HLA class I peptide epitope supermotifs and motifs aresummarized below. In some cases, peptides may be listed in both a motifand a supermotif summary. The relationship of a particular motif andrespective supermotif is indicated in the description of the individualmotifs.

i. HLA-A1 Supermotif

The HLA-A1 supermotif is characterized by the presence in peptideligands of a small (T or S) or hydrophobic (L, I, V, or M) primaryanchor residue in position 2, and an aromatic (Y, F, or W) primaryanchor residue at the C-terminal position of the epitope. Thecorresponding family of HLA molecules that bind to the A1 supermotif(i.e., the HLA-A1 supertype) is comprised of at least A*0101, A*2601,A*2602, A*2501, and A*3201 (see, e.g., DiBrino, M. et al., J. Immunol.151:5930, 1993; DiBrino, M. et al., J. Immunol. 152:620, 1994; Kondo, A.et al., Immunogenetics 45:249, 1997).

ii. HLA-A2 Supermotif

Primary anchor specificities for allele-specific HLA-A2.1 molecules(see, e.g., Falk et al., Nature 351:290-296, 1991; Hunt et al., Science255:1261-1263, 1992; Parker et al., J. Immunol. 149:3580-3587, 1992;Ruppert et al., Cell 74:929-937, 1993) and cross-reactive binding amongHLA-A2 and -A28 molecules have been described. (See, e.g., Fruci et al.,Human Immunol. 38:187-192, 1993; Tanigaki et al., Human Immunol.39:155-162, 1994; Del Guercio et al., J. Immunol. 154:685-693, 1995;Kast et al., J. Immunol. 152:3904-3912, 1994 for reviews of relevantdata.) These primary anchor residues define the HLA-A2 supermotif; whichpresence in peptide ligands corresponds to the ability to bind severaldifferent HLA-A2 and -A28 molecules. The HLA-A2 supermotif comprisespeptide ligands with L, I, V, M, A, T, or Q as a primary anchor residueat position 2 and L, I, V, M, A, or T as a primary anchor residue at theC-terminal position of the epitope.

The corresponding family of HLA molecules (i.e., the HLA-A2 supertypethat binds these peptides) is comprised of at least: A*0201, A*0202,A*0203, A*0204, A*0205, A*0206, A*0207, A*0209, A*0214, A*6802, andA*6901.

iii. HLA-A3 Supermotif

The HLA-A3 supermotif is characterized by the presence in peptideligands of A, L, I, V, M, S, or T as a primary anchor at position 2, anda positively charged residue, R or K, at the C-terminal position of theepitope, e.g., in position 9 of 9-mers (see, e.g., Sidney et al., Hum.Immunol. 45:79, 1996). Exemplary members of the corresponding family ofHLA molecules (the HLA-A3 supertype) that bind the A3 supermotif includeat least A*0301, A*1101, A*3101, A*3301, and A*6801.

iv. HLA-A24 Supermotif

The HLA-A24 supermotif is characterized by the presence in peptideligands of an aromatic (F, W, or Y) or hydrophobic aliphatic (L, I, V,M, or T) residue as a primary anchor in position 2, and Y, F, W, L, I,or M as primary anchor at the C-terminal position of the epitope (see,e.g., Sette and Sidney, Immunogenetics, 50:201-212, 1999). Thecorresponding family of HLA molecules that bind to the A24 supermotif(i.e., the A24 supertype) includes at least A*2402, A*3001, and A*2301.

v. HLA-B7 Supermotif

The HLA-B7 supermotif is characterized by peptides bearing proline inposition 2 as a primary anchor, and a hydrophobic or aliphatic aminoacid (L, I, V, M, A, F, W, or Y) as the primary anchor at the C-terminalposition of the epitope. The corresponding family of HLA molecules thatbind the B7 supermotif (i.e., the HLA-B7 supertype) is comprised of atleast twenty six HLA-B proteins including: B*0702, B*0703, B*0704,B*0705, B*1508, B*3501, B*3502, B*3503, B*3504, B*3505, B*3506, B*3507,B*3508, B*5101, B*5102, B*5103, B*5104, B*5105, B*5301, B*5401, B*5501,B*5502, B*5601, B*5602, B*6701, and B*7801 (see, e.g., Sidney, et al.,J. Immunol. 154:247, 1995; Barber, et al., Curr. Biol. 5:179, 1995;Hill, et al., Nature 360:434, 1992; Rammensee, et al., Immunogenetics41:178, 1995 for reviews of relevant data).

vi. HLA-B27 Supermotif

The HLA-B27 supermotif is characterized by the presence in peptideligands of a positively charged (R, H, or K) residue as a primary anchorat position 2, and a hydrophobic (F, Y, L, W, M, I, A, or V) residue asa primary anchor at the C-terminal position of the epitope (see, e.g.,Sette and Sidney, Immunogenetics, 50:201-212, 1999). Exemplary membersof the corresponding family of HLA molecules that bind to the B27supermotif (i.e., the B27 supertype) include at least B*1401, B*1402,B*1509, B*2702, B*2703, B*2704, B*2705, B*2706, B*3801, B*3901, B*3902,and B*7301.

vii. HLA-B44 Supermotif

The HLA-B44 supermotif is characterized by the presence in peptideligands of negatively charged (D or E) residues as a primary anchor inposition 2, and hydrophobic residues (F, W, Y, L, I, M, V, or A) as aprimary anchor at the C-terminal position of the epitope (see, e.g.,Sidney et al., Immunol. Today 17:261, 1996). Exemplary members of thecorresponding family of HLA molecules that bind to the B44 supermotif(i.e., the B44 supertype) include at least: B*1801, B*1802, B*3701,B*4001, B*4002, B*4006, B*4402, B*4403, and B*4006.

viii. HLA-B58 Supermotif

The HLA-B58 supermotif is characterized by the presence in peptideligands of a small aliphatic residue (A, S, or T) as a primary anchorresidue at position 2, and an aromatic or hydrophobic residue (F, W, Y,L, I, V, M, or A) as a primary anchor residue at the C-terminal positionof the epitope (see, e.g., Sette and Sidney, Immunogenetics, 50:201-212,1999 for reviews of relevant data). Exemplary members of thecorresponding family of HLA molecules that bind to the B58 supermotif(i.e., the B58 supertype) include at least: B*1516, B*1517, B*5701,B*5702, and B*5801.

ix. HLA-B62 Supermotif

The HLA-B62 supermotif is characterized by the presence in peptideligands of the polar aliphatic residue Q or a hydrophobic aliphaticresidue (L, V, M, I, or P) as a primary anchor in position 2, and ahydrophobic residue (F, W, Y, M, I, V, L, or A) as a primary anchor atthe C-terminal position of the epitope (see, e.g., Sette and Sidney,Immunogenetics, 50:201-212, 1999). Exemplary members of thecorresponding family of HLA molecules that bind to the B62 supermotif(i.e., the B62 supertype) include at least: B*1501, B*1502, B*1513, andB5201.

x. HLA-A1 Motif

The HLA-A1 motif is characterized by the presence in peptide ligands ofT, S, or M as a primary anchor residue at position 2 and the presence ofY as a primary anchor residue at the C-terminal position of the epitope.An alternative allele-specific A1 motif is characterized by a primaryanchor residue at position 3 rather than position 2. This motif ischaracterized by the presence of D, E, A, or S as a primary anchorresidue in position 3, and a Y as a primary anchor residue at theC-terminal position of the epitope (see, e.g., DiBrino et al., J.Immunol., 152:620, 1994; Kondo et al., Immunogenetics 45:249, 1997; andKubo et al., J. Immunol. 152:3913, 1994 for reviews of relevant data).

xi. HLA-A*0201 Motif

An HLA-A2*0201 motif was determined to be characterized by the presencein peptide ligands of L or M as a primary anchor residue in position 2,and L or V as a primary anchor residue at the C-terminal position of a9-residue peptide (see, e.g., Falk et al., Nature 351:290-296, 1991) andwas further found to comprise an I at position 2 and I or A at theC-terminal position of a nine amino acid peptide (see, e.g., Hunt etal., Science 255:1261-1263, Mar. 6, 1992; Parker et al., J. Immunol.149:3580-3587, 1992). The A*0201 allele-specific motif has also beendefined to additionally comprise V, A, T, or Q as a primary anchorresidue at position 2, and M or T as a primary anchor residue at theC-terminal position of the epitope (see, e.g., Kast et al., J. Immunol.152:3904-3912, 1994). Thus, the HLA-A*0201 motif comprises peptideligands with L, I, V, M, A, T, or Q as primary anchor residues atposition 2 and L, I, V, M, A, or T as a primary anchor residue at theC-terminal position of the epitope. The preferred and tolerated residuesthat characterize the primary anchor positions of the HLA-A*0201 motifare identical to the residues describing the A2 supermotif.

xii. HLA-A3 Motif

The HLA-A3 motif is characterized by the presence in peptide ligands ofL, M, V, I, S, A, T, F, C, G, or D as a primary anchor residue atposition 2, and the presence of K, Y, R, H, F, or A as a primary anchorresidue at the C-terminal position of the epitope (see, e.g., DiBrino etal., Proc. Natl. Acad. Sci USA 90:1508, 1993; and Kubo et al., J.Immunol. 152:3913-3924, 1994).

xiii. HLA-A1 Motif

The HLA-A11 motif is characterized by the presence in peptide ligands ofV, T, M, L, I, S, A, G, N, C, D, or F as a primary anchor residue inposition 2, and K, R, Y, or H as a primary anchor residue at theC-terminal position of the epitope (see, e.g., Zhang et al., Proc. Natl.Acad. Sci USA 90:2217-2221, 1993; and Kubo et al., J. Immunol.152:3913-3924, 1994).

xiv. HLA-A24 Motif

The HLA-A24 motif is characterized by the presence in peptide ligands ofY, F, W, or M as a primary anchor residue in position 2, and F, L, I, orW as a primary anchor residue at the C-terminal position of the epitope(see, e.g., Kondo et al., J. Immunol. 155:4307-4312, 1995; and Kubo etal., J. Immunol. 152:3913-3924, 1994).

Parent peptides may be from any target antigen. Preferred targetantigens are tumor-associated (TAA) or tumor-specific antigens (TSA) andantigens produced by an infectious agent or a host protein producedduring infection. The term “target antigen” refers to any protein orpeptide that, when introduced into a host having an immune system(directly or upon expression as in, e. g., DNA vaccines), is recognizedby the immune system of the host and is capable of eliciting a specificimmune reaction. The terms “tumor-associated antigen (TAA)” and“tumor-specific antigen (TSA)” are used interchangeably and refer to anantigenic peptide that is associated with a tumor or a cancerous cell.TAAs include, for example, mutated cellular proteins such as mutatedtumor suppressor gene products, oncogene products (including fusionproteins), and foreign proteins such as viral gene products. Non-mutatedcellular proteins may also be TAAs if they are expressed aberrantly (e.g., in an inappropriate subcellular compartment) or in supernormalquantities.

Examples of preferred target antigens include TAAs such as ErbBreceptors, Melan A (MART1), gp100, tyrosinase, TRP-1/gp 75, and TRP-2(in melanoma; for additional examples, see also a list of antigensprovided in Storkus and Zarour, Forum (Genova), 2000 July-September, 10(3): 256-270); MAGE-1 and MAGE-3 (in bladder, head and neck, andnon-small cell carcinoma); HPV EG and E7 proteins (in cervical cancer);Mucin (MUC-1) (in breast, pancreas, colon, and prostate cancers);prostate-specific antigen (PSA) (in prostate cancer); carcinoembryonicantigen (CEA) (in colon, breast, lung, thyroid, and gastrointestinalcancers), P1A tumor antigen (e. g., as disclosed in International PatentPublication No. WO 98/56919), and such shared tumor-specific antigens asMAGE-2, MAGE-4, MAGE-6, MAGE-10, MAGE-12, BAGE-1, CAGE-1, 2, 8, CAGE-3to 7, LAGE-1, NY-ESO-1/LAGE-2, NA-88, and GnTV (see, e. g.,International Patent Publication No. WO 98/56919).

In a specific embodiment, the target antigen is carcinoembryonic antigen(CEA). CEA is associated with neoplasms of epithelial origin, includingcarcinomas of the gastrointestinal tract, breast, lung, and thyroid, andtherefore, constructs of the invention that include CEA epitopes may beused to threat neoplasms of epithelial origin.

Other target antigens of the invention include but are not limited to(i) protozoan antigens such as those derived from Plasmodium spp.,Toxoplasma spp., Pneumocystis carinii, Leishmania sp., and Trypanosomaspp.; (ii) viral protein or peptide antigens such as those derived frominfluenza virus (e. g., surface glycoproteins hemagluttinin (HA) andneuraminidase (NA) or the nucleoprotein (NP) as described in Bodmer etal., Cell, 52: 253, 1988 and Tsuji et al., J. Virol. 72:6907-6910, 1998or NP CTL epitopes as described in Gould et al., J. Virol., 65:5401,1991; Murata et al., Cell Immunol., 173:96-107, 1996, and PCTApplication No. WO 98/56919); immunodeficiency virus, e. g., a simianimmunodeficiency virus (SIV) antigen (e. g., SIV-env CTL epitope asdisclosed in PCT Application No. WO 98/56919), or a humanimmunodeficiency virus antigen (HIV-1) such as gp120 CTL epitopes asdisclosed, e. g., in PCT Application No. WO 98/56919, gp160, pl8 antigen(e. g., CD8+ T cell epitopes and gp41 CTL epitopes as disclosed, e. g.,in PCT Application No. WO 98/56919, Gag p24 CD8+ T cell epitopes, Gagpl7 CD8+ T cell epitopes, Tat, Pol, Nef CTL epitopes as disclosed, e.g., in PCT Application No. WO 98/56919, and Env CTL epitopes asdisclosed, e. g., in PCT Application No. WO 98/56919); herpesvirus, e.g., a glycoprotein, for instance, from feline herpesvirus, equineherpesvirus, bovine herpesvirus, pseudorabies virus, canine herpesvirus,herpes simplex virus (HSV, e. g., HSV tk, gB, gD), herpes zoster virus,Marek's Disease Virus, herpesvirus of turkeys (HVT), cytomegalovirus(CMV), or Epstein-Barr virus; hepatitis C virus; human papilloma virus(HPV); human T cell leukemia virus (HTLV-1); bovine leukemia virus (e.g., gp51, 30 envelope antigen); feline leukemia virus (FeLV) (e. g.,FeLV envelope protein, a Newcastle Disease Virus (NDV) antigen, e. g.,HN or F); rous associated virus (such as RAV-1 env); infectiousbronchitis virus (e. g., matrix and/or preplomer); flavivirus (e. g., aJapanese encephalitis virus (JEV) antigen, a Yellow Fever antigen, or aDengue virus antigen); Morbillivirus (e. g., a canine distemper virusantigen, a measles antigen, or rinderpest antigen such as HA or F);rabies (e. g., rabies glycoprotein G); parvovirus (e. g., a canineparvovirus antigen); hepatitis C virus (HCV); poxvirus (e. g., anectromelia antigen, a canary poxvirus antigen, or a fowl poxvirusantigen such as chicken pox virus varicella zoster antigen); infectiousbursal disease virus (e. g., VP2, VP3, or VP4); Hantaan virus; mumpsvirus, and measles virus; (iii) bacterial antigens such as Mycobacteriumtuberculosis-specific (e. g., Bacillus Calmette-Guerin (BCG)-38 kDprotein; antigen 85 complex (as described in Klein et al., J. Infect.Dis., 183:928-34, 2001), see also a list of antigens in Klein andMcAdam, Arch. Immunol. Ther. Exp. (Warsz.), 47: 313-320, 1999), Listeriamonocytoges-specific (e. g., as disclosed in Finelli et al., Immunol.Res., 19: 211-223, 1999), Salmonella typhii-specific, Shigellaflexineri-specific, staphylococcus-specific, streptococcus-specific,pneumococcus-specific (e. g., PspA (see PCT Publication No. WO92/14488)), Neisseria gonorrhea-specific, Borelia-specific (e. g., OspA,OspB, OspC antigens of Borrelia associated with Lyme disease such asBorrelia bergdorferi, Borrelia afzelli, and Borrelia garinii (see, e.g.,U.S. Pat. No. 5,523,089; PCT Application Nos. WO 90/04411, WO 91/09870,WO 93/04175, WO 96/06165, WO93/08306; PCT/US92/08697; Bergstrom et al.,Mol. Microbiol., 3:479-486, 1989; Johnson et al., Infect. and Immun.60:1845-1853, 1992; Johnson et al., Vaccine 13:1086-1094, 1995; TheSixth International Conference on Lyme Borreliosis: Progress on theDevelopment of Lyme Disease Vaccine, Vaccine 13:133-135, 1995)), A.pertussis-specific, S. parathyphoid A and B-specific, C.diphtheriae-specific, C. tetanus-specific, C. botulinum-specific, C.perfringens-specific, A. anthracis-specific, A. pestis-specific, Vcholera-specific, H. influenzae-specific, T. palladium-specific,Chlamydia trachomatis-specific (e. g., as disclosed in Kim et al., J.Immunol., 162: 6855-6866, 1999), and pseudomonas-specific proteins orpeptides; and (iv) fungal antigens such as those isolated from candida(e. g., 65 kDa mannoprotein (MP65) from Candida albicans), trichophyton,or pityrosporum.

The foregoing list of target antigens is intended as exemplary, as theantigen of interest can be derived from any animal or human pathogen ortumor or cancerous cell. With respect to DNA encoding pathogen-derivedantigens of interest, attention is directed to, e. g., U.S. Pat. Nos.4,722,848; 5,174,993; 5,338,683; 5,494,807; 5,503,834; 5,505,941;5,514,375; 5,529,780; U. K. Patent No. GB 2 269 820 B; and PCTPublication Nos. WO 92/22641; WO 93/03145; WO 94/16716; WO 96/3941;PCT/US94/06652. With respect to antigens derived from tumor viruses,reference is also made to Molecular Biology of Tumor Viruses, RNA TumorViruses, Second Edition, Edited by Weiss et al., Cold Spring HarborLaboratory Press, 1982. For a list of additional antigens useful in thecompositions of the invention see also Stedman's Medical Dictionary(24th edition, 1982).

Additional examples of target antigens include prostate specificantigens (PSA), melanoma antigens MAGE-1, MAGE-2, MAGE-3, MAGE-11,MAGE-A10, as well as BAGE, GAGE, RAGE, MAGE-C1, LAGE-1, CAG-3, DAM,MUC1, MUC2, MUC18, NY-ESO-1, MUM-1, CDK4, BRCA2, NY-LU-1, NY-LU-7,NY-LU-12, CASP8, RAS, KIAA-2-5, SCCs, p53, p73, CEA, Her 2/neu, Melan-A,gp100, tyrosinase, TRP2, gp75/TRP1, kallikrein, prostate-specificmembrane antigen (PSM), prostatic acid phosphatase (PAP),prostate-specific antigen (PSA), PT1-1, β-catenin, PRAME, Telomerase,FAK, cyclin D1 protein, NOEY2, EGF-R, SART-1, CAPB, HPVE7, p15, Folatereceptor CDC27, PAGE-1, and PAGE-4.

Additional examples of target antigens include hepatitis B core andsurface antigens (HBVc, HBVs), hepatitis C antigens, Epstein-Barr virusantigens, human immunodeficiency virus (HIV) antigens and humanpapilloma virus (HPV) antigens, Mycobacterium tuberculosis andChlamydia. Examples of suitable fungal antigens include those derivedfrom Candida albicans, Cryptococcus neoformans, Coccidoides spp.,Histoplasma spp, and Aspergillus fumigatis. Examples of suitableprotozoan parasitic antigens include those derived from Plasmodium spp.,including P. falciparum, Trypanosoma spp., Schistosoma spp., Leishmaniaspp and the like.

In a non-limiting embodiment, the parent peptide from which a peptidederivative of the present invention can be prepared is selected from thegroup consisting of an HLA-A0201-restricted peptide from a universaltumor antigen. In a non-limiting embodiment, the parent peptide isselected from Survivin, hTERT and CYP1B1. In another non-limitingembodiment, the parent peptide is an HLA-A0201-restricted MART-1peptide.

In a non-limiting embodiment, peptide derivatives provided by thepresent invention are based on the MART-1 peptide epitope of Melan-A,the amino acid sequence of which is AAGIGILTV (SEQ ID NO:1). Such aMART-1-based peptide derivative can be based on the 9-mer (nonamer)parent amino acid sequence AAGIGILTV (SEQ ID NO:1); or on the 10-mer(decamer) parent amino acid sequence EAAGIGILTV (SEQ ID NO:2).

In one non-limiting embodiment, the present invention provides peptidederivatives based on the parent or wild type MART-1 nonamer sequenceAAGIGILTV (SEQ ID NO:1), wherein the peptide derivatives have asubstitution at a position corresponding to amino acid Position 2 (P2)in the nonamer peptide (as numbered from the amino terminus of thenonamer peptide), such that the alanine residue present at P2 of thewild type nonamer peptide is replaced with a non-natural amino acid.Such “nonamer” peptide derivatives of the present invention have thegeneral formula A-X_(aa)-GIGILTV (SEQ ID NO:3), where X_(aa) representsa non-natural amino acid. In one embodiment, the non-natural amino acidhas increased conformational constraint compared to the conformationalconstraint present in the original amino acid at P2 in parent peptideAAGIGILTV (SEQ ID NO:1). In some embodiments, the derivative peptidethat results from the introduction of the non-natural amino acid has anincreased conformational constraint compared to the parent peptide. Suchsubstitution at P2 of this wild type nonamer sequence serves to provide“MART-1-based peptide derivatives” having at least one, or at least two,or at least three, or at least four, or all five, of the aforementionedproperties.

Specific non-limiting embodiments of such MART-1-based peptidederivatives include:

(a)  (SEQ ID NO: 4) A-[c3a]-GIGILTV; (b)  (SEQ ID NO: 5)A-[c5g]-GIGILTV; (c)  (SEQ ID NO: 6) A-[chg]-GIGILTV; (d) (SEQ ID NO: 7) A-[cpg]-GIGILTV; (e)  (SEQ ID NO: 8) A-[dfb]-GIGILTV;(f)  (SEQ ID NO: 9) A-[dhl]-GIGILTV; (g)  (SEQ ID NO: 10)A-[phg]-GIGILTV;  and (h)  (SEQ ID NO: 11) A-[sta]-GIGILTV.

In another non-limiting embodiment, the present invention providespeptide derivatives based on the parent or wild type MART-1 decamersequence EAAGIGILTV (SEQ ID NO:2), wherein the peptide derivatives havea substitution at a position corresponding to amino acid Position 2 (P2)in the decamer peptide (as numbered from the amino terminus of thedecamer peptide), such that the alanine residue present at P2 of thewild type decamer peptide is replaced with a non-natural amino acid.Such “decamer” peptide derivatives of the present invention have thegeneral formula E-X_(aa)-AGIGILTV (SEQ ID NO:12), where X_(aa)represents a non-natural amino acid. In some embodiments, thenon-natural amino acid has increased conformational constraint in thepeptide derivative compared to the conformational constraint of theoriginal amino acid in the wild type decamer peptide EAAGIGILTV (SEQ IDNO:2). In some embodiments, the derivative peptide that results from theintroduction of the non-natural amino acid has an increasedconformational constraint compared to the parent peptide. Suchsubstitution at P2 of this wild type decamer sequence thus serves toprovide additional “MART-1-based peptide derivatives” having at leastone, or at least two, or at least three, or at least four, or all fiveof the aforementioned properties.

Specific non-limiting embodiments of such MART-1-based peptidederivatives include:

(a)  (SEQ ID NO: 13) E-[c3a]-AGIGILTV; (b)  (SEQ ID NO: 14)E-[c5g]-AGIGILTV; (c)  (SEQ ID NO: 15) E-[chg]-AGIGILTV; (d) (SEQ ID NO: 16) E-[cpg]-AGIGILTV; (e)  (SEQ ID NO: 17) E-[dfb]-AGIGILTV;(f)  (SEQ ID NO: 18) E-[dhl]-AGIGILTV; (g)  (SEQ ID NO: 19)E-[phg]-AGIGILTV;  and (h)  (SEQ ID NO: 20) E-[sta]-AGIGILTV.

In another non-limiting embodiment, peptide derivatives provided by thepresent invention are based on a peptide selected from the Survivinprotein (GenBank #U75285; Reed, J. C., 2001, J. Clin. Invest. 108:965-969). The Survivin protein is a member of the inhibitors ofapoptosis gene family, which suppresses apoptosis and regulates celldivision (Reed, J. C., 2001, J. Clin. Invest. 108: 965-969). TheSurvivin protein appears to preferentially inhibitmitochondrial-dependent apoptosis by targeting caspase-9 and plays acritical role in mitosis and embryonic development. Multiple splicevariants of Survivin have been identified (Mahotka, C, et al, 1999,Cancer Res. 59, 6097-6102; Badran, A, et al, 2004, Biochem. Biophys.Res. Commun. 314, 902-907), but the functional significance of thesevariants has not been established. Survivin is among the mosttumor-specific of all human gene products and over-expression has beendocumented in many of the major tumor types. In addition,over-expression of Survivin in cancer patients has been correlated witha more aggressive disease and poor survival.

In one non-limiting embodiment, peptide derivatives of the presentinvention are based on the parent or wild type Survivin nonamer sequenceISTFKNWPF (SEQ ID NO:21), wherein the peptide derivatives have asubstitution at amino acid Position 2 (P2) (as numbered from the aminoterminus of the nonamer peptide), such that the serine residue presentat position P2 of the wild type nonamer peptide is replaced with anon-natural amino acid. Such “nonamer” peptide derivatives have thegeneral formula I-X_(aa)-TFKNWPF (SEQ ID NO:22), where X_(aa) is anon-natural amino acid. In some embodiments, the non-natural amino acidhas increased conformational constraint in the peptide derivativecompared to the conformational constraint of the original amino acid inthe nonamer peptide ISTFKNWPF (SEQ ID NO:21). In some embodiments, thederivative peptide that results from the introduction of the non-naturalamino acid has an increased conformational constraint compared to theparent peptide. Such substitution at P2 of this wild type nonamersequence serves to provide “Survivin-based peptide derivatives” havingat least one, or at least two, or at least three, or at least four, orall five of the aforementioned properties.

Specific non-limiting embodiments of such Survivin-based peptidederivatives include:

(a)  (SEQ ID NO: 23) I-[c3a]-TFKNWPF; (b)  (SEQ ID NO: 24)I-[c5g]-TFKNWPF; (c)  (SEQ ID NO: 25) I-[chg]-TFKNWPF; (d) (SEQ ID NO: 26) I-[cpg]-TFKNWPF; (e)  (SEQ ID NO: 27) I-[dfb]-TFKNWPF;(f)  (SEQ ID NO: 28) I-[dhl]-TFKNWPF; (g)  (SEQ ID NO: 29)I-[phg]-TFKNWPF;  and (h)  (SEQ ID NO: 30) I-[sta]-TFKNWPF.

In another non-limiting embodiment, the present invention providespeptide derivatives based on the wild type Survivin nonamer sequence ofKVRRAIEQL (SEQ ID NO:31), wherein the peptide derivatives have asubstitution at a position corresponding to amino acid Position 2 (P2)in this nonamer peptide (as numbered from the amino terminus of thedecamer peptide), such that the valine residue present at P2 of the wildtype nonamer peptide is replaced with a non-natural amino acid. Such“nonamer” peptide derivatives of the present invention have the generalformula K-X_(aa)-RRAIEQL (SEQ ID NO:32), where X_(aa) represents anon-natural amino acid. In some embodiments, the non-natural amino acidhas increased conformational constraint in the peptide derivativecompared to the conformational constraint present in the original aminoacid in the wild type nonamer peptide KVRRAIEQL (SEQ ID NO:31). In someembodiments, the derivative peptide that results from the introductionof the non-natural amino acid has an increased conformational constraintcompared to the parent peptide. Such substitution at P2 of this wildtype nonamer sequence serves to provide additional “Survivin-basedpeptide derivatives” having at least one, or at least two, or at leastthree, or at least four, or all five of the aforementioned properties.

Specific non-limiting embodiments of such Survivin-based peptidederivatives include:

(a)  (SEQ ID NO: 33) K-[c3a]-RRAIEQL; (b)  (SEQ ID NO: 34)K-[c5g]-RRAIEQL; (c)  (SEQ ID NO: 35) K-[chg]-RRAIEQL; (d) (SEQ ID NO: 36) K-[cpg]-RRAIEQL; (e)  (SEQ ID NO: 37) K-[dfb]-RRAIEQL;(f)  (SEQ ID NO: 38) K-[dhl]-RRAIEQL; (g)  (SEQ ID NO: 39)K-[phg]-RRAIEQL;  and (h)  (SEQ ID NO: 40) K-[sta]-RRAIEQL.

Any peptide derivative provided by the present invention can optionallyfurther comprise one or more other modifications so as to: (i) furtherimprove one, or two, or three, or four, or all five of theaforementioned properties obtained by the single substitution alone asdescribed above and/or (ii) improve a different property than the one ormore of the aforementioned properties obtained by the singlesubstitution alone as described above. For example, the peptidederivative prepared as described above can further comprise asubstitution of a second naturally occurring amino acid residue in theparent peptide with the same or different non-natural amino acid as usedin the first substitution.

In a non-limiting example, a peptide derivative of the present inventioncan incorporate a second substitution. Such a second substitution may beat a second primary anchor position or another position in the parentpeptide. Such a second substitution may be at Position 6 (P6), atPosition 7 (P7), or at Position Omega (PΩ), which is the amino acidresidue occupying the last position at the carboxyl end of the peptide.As with the first amino acid substitution described above, the secondamino acid substitution can serve to replace a naturally occurring aminoacid residue in the peptide with a non-natural amino acid, such as aconformation constraining non-natural amino acid residue. For example,the amino acid residue at PΩ in the peptide can be substituted with aconformation constraining non-natural amino acid residue to furtherincrease the conformational constraint within the peptide derivative.

In a non-limiting embodiment, a general formula for a peptide derivativeof the present invention based on a nonamer MART-1 peptide and having adouble substitution is A-X_(aa1)-GIG-X_(aa2)-LTV (SEQ ID NO:41).

In another non-limiting embodiment, a general formula for a peptidederivative of the present invention based on a nonamer MART-1 peptideand having a double substitution is A-X_(aa1)GIGI-LT-X_(aa2) (SEQ IDNO:42).

In another non-limiting embodiment, a general formula for a peptidederivative of the present invention based on a decamer MART-1 peptideand having a double substitution is E-X_(aa1)-AGIG-X_(aa2)-LTV (SEQ IDNO:43), in a specific embodiment it is E-[c5g]-AGIG-[amv]-LTV (SEQ IDNO:49).

In another non-limiting embodiment, a general formula for a peptidederivative of the present invention based on a decamer MART-1 peptideand having a double substitution is E-X_(aa1)-AGIGILT-X_(aa2) (SEQ IDNO:44).

In another non-limiting embodiment, a general formula for a peptidederivative of the present invention based on a nonamer Survivin peptidesequence and having a double substitution is I-X_(aa1)TFK-X_(aa2)-WPF(SEQ ID NO:45).

In another non-limiting embodiment, a general formula for a peptidederivative of the present invention based on a nonamer Survivin sequenceand having a double substitution is I-X_(aa1)-TFKNWP-X_(aa2) (SEQ IDNO:46).

In another non-limiting embodiment, a general formula for a peptidederivative of the present invention based on a nonamer Survivin peptideand having a double substitution is K-X_(aa1)-RRA-X_(aa2)-EQL (SEQ IDNO:47).

In another non-limiting embodiment, a general formula for a peptidederivative of the present invention based on a nonamer Survivin peptideand having a double substitution is K-X_(aa1)-RRAI-EQ-X_(aa2) (SEQ IDNO:48).

In each of the above cases, each of X_(aa1) and X_(aa2) is independentlyselected from non-natural amino acids such as those that can provideincreased conformational constraint in the particular peptide comparedto the conformational constraint present in the parent peptide.

A peptide derivative of the invention may comprise additional changesfrom a wild type peptide. Such changes include replacement of a primaryor secondary anchor residue with a conserved or semi-conserved residueat that position. Likewise, a peptide derivative of the invention mayhave a replacement of an amino acid at a non-anchor position withanother amino acid, as described in US 20060018915 A2 to form aheteroclitic analog. Combinations of such replacements may be made inthe wild type peptide to form an analog. Then, one or more amino acidsof the analog are replaced with a non-natural amino acid to form apeptide derivative of the invention. Alternatively, a peptide derivativemay be made that only contains the non-natural amino acid(s) of theinvention, and then further modifications are made in the peptidederivative to form a peptide derivative containing, e.g., a conservativeor semi-conservative substitution at an anchor position or aheteroclitic substitution.

Parent peptides and peptide derivatives of the present invention can besynthesized by any method known in the art of protein chemistry. Thepresent invention does not require that a peptide derivative of thepresent invention be synthesized directly from a parent peptide assubstrate to be converted to the peptide derivative. Instead, a peptidederivative of the present invention will most conveniently be preparedby de novo synthesis using, e.g., a commercially available peptidesynthesizer.

The term “subject” as used herein refers to an animal having an immunesystem, preferably a mammal such as a rodent (e.g., mouse or rat), acompanion animal (dog or cat), or a primate, and particularly a human.

The terms “therapeutically effective amount” and “therapeuticallyeffective dose” refer to that quantity of a peptide derivative orpharmaceutical composition of the present invention that is sufficientto induce an immune response upon administration to a subject in needthereof or upon contacting a cell(s). As used herein with respect topharmaceutical compositions, the terms “therapeutically effectiveamount” and “therapeutically effective dose” (used interchangeably withthe term “immunogenetically effective amount” or “immunogeneticallyeffective dose”) refer to the amount or dose of a peptide derivative orpharmaceutical composition of the present invention sufficient toproduce an effective immune response upon administration to a subject orupon contacting a cell(s).

For methods of treating a cancer, a “therapeutically effective dose” isan amount or dose of the peptide derivative or pharmaceuticalcomposition of the present invention sufficient to produce ananti-cancer immune response in vivo useful to slow or reverse theprogression of the cancer, or to reduce the severity of at least onesymptom of the cancer.

For methods of preventing a cancer, a “therapeutically effective dose”is an amount or dose of the peptide derivative or pharmaceuticalcomposition of the present invention sufficient to produce ananti-cancer immune response in vivo useful to prevent the onset of thecancer in, e.g., a subject prone to developing such a cancer asdetermined, e.g., by a diagnostic test or in view of a prior familyhistory of cancer.

For methods of treating a pathogen-specific infection, a“therapeutically effective dose” is an amount or dose of the peptidederivative or pharmaceutical composition of the present inventionsufficient to produce an immune response in vivo against that pathogen,which immune response is useful to slow or reverse the progression ofthe infection.

For methods of preventing a pathogen-specific infection, a“therapeutically effective dose” is an amount or dose of the peptidederivative or pharmaceutical composition of the present inventionsufficient to produce an immune response in vivo against that pathogen,which immune response is useful to prevent the infection or preemptivelyto reduce the severity of infection produced by the pathogen.

4.2. Approach

The present invention provides a peptide derivative that is an MHC classI (“MHCI”) restricted antigen, and which is prepared or derived from aparent peptide of 8 to 11 amino acid residues in length, and preferablyeither 9 or 10 amino acid residues in length, wherein the peptidederivative contains a non-natural amino acid substituted in place of anaturally occurring amino acid residue at one or more primary anchorpositions, preferably at two primary anchor positions, and morepreferably at one primary anchor position. Thus, the invention providesa peptide derivative of a MHCI restricted parent antigen (e.g., parentpeptide or parent epitope) comprising a non-natural amino acid in atleast one anchor position.

The invention further provides a peptide derivative of a MHCI restrictedparent antigen comprising a non-natural amino acid at one anchorposition and a second substitution at a second position. The secondsubstitution at a second position may be at a second anchor position, ormay be at the C-terminus (PΩ), e.g., for MHCI-peptides that do not havean anchor position at the C-terminus, or may be at position 6 (P6) orposition 7 (P7).

A goal of the present invention is to create a streamlined, rationalprocess by which to modify parent peptides (naturally occurring (i.e.,wild type) peptide epitopes and relevant analogs) to create novelmodified peptide molecules (peptide derivatives) that will moreeffectively stimulate the immune system to actively recognize anddestroy cancer cells. These peptide derivatives are useful in certainpharmaceutical compositions and methods of the present invention totreat or prevent a cancer in a subject.

This rational process can also be used to modify parent peptides (wildtype peptide epitopes and other relevant parent peptides) to createnovel peptide derivatives that will stimulate the immune system toactively recognize and destroy infected cells. These peptide derivativesare useful in certain pharmaceutical compositions and methods of thepresent invention to treat or prevent an infection in a subject.

Previous methods for selecting other types of improved peptide epitopesutilized either: (i) prediction algorithms, which scan the targetprotein and predict which peptides would be epitopes based on thepresence of specific residues at specific positions; or (ii) naturallyimmunized T-cell populations and peptide libraries to identify epitopesby directly testing for naturally occurring reactive T-cells.

A central aspect of the present invention is the replacement of at leastone amino acid residue in an anchor position in the parent peptide witha non-natural amino acid. In some embodiments, the non-natural aminoacid introduces increased conformational constraint into the peptidemolecule. The present invention achieves peptide derivatives withimproved immunogenicity, via, e.g., improved binding affinity or slowingof the kinetic off-rate from an MHCI (e.g., HLA-A2). Unlike approachesbased on the high throughput screening of thousands or millions ofcompounds, the present invention typically provides optimized antigenicsequences by synthesizing less than 100 total compounds for any givenparent sequence.

To demonstrate the effectiveness of this approach, several peptidederivatives were prepared by substituting one of several differentnon-natural amino acid residues into an anchor position of the MART-1peptide, and these were tested using HLA-A0201, as described in theExamples below. Each non-natural amino acid substituted into the anchorpositions was selected to impose a conformational constraint in theresulting peptide, leading to enhanced binding affinity of the peptidederivative to MHCI and increased stability of the MHCI-peptidederivative complex. These peptide derivatives were more potent than thewild type MART-1 peptide at inducing a CTL response, and displayedenhanced activity in T-cell assays. When used in in vitro education(IVE) experiments, the peptide derivatives are more efficient inexpanding peptide specific T-cells, and the expanded T-cells recognizethe wild type sequence, as demonstrated by ELISPOT assays. Such expandedT-cells would also be expected to recognize the wild type sequence inCTL lysis assays that are known in the art. Additionally,peptide-binding assays have been used to directly measure the affinitiesand off-rates of the peptide derivatives, demonstrating that thesebinding parameters can be selectively influenced. Thus, through thesynthesis of fewer than 40 conformationally constrained peptides,several peptide derivatives based on the MART-1 epitope have beenidentified displaying higher affinity and similar or superiorimmunogenicity to the altered peptide ligand (“APL”) MART-1 peptide.Using the same approach, novel peptide derivatives based on the Survivinprotein have also been identified, as disclosed herein.

The present invention is thus based on two unexpected results. Firstly,the replacement of the consensus amino acid at an anchor position of thepeptide (e.g., replacement of Leu at P2 of an HLA-A0201-restrictedpeptide) by a non-natural amino acid (such as an appropriateconformation constraining non-natural amino acid) can result in anincrease in MHC-peptide affinity. Secondly, such increased affinity canresult in increased immunogenicity of the peptide derivative compared tothe wild type peptide in T-cell assays such as T-cell proliferation,cytokine production and cell lysis assays or other assays describedherein or known in the art.

To identify a suitable parent peptide, a number of methods may be used,such as computational methods like SYFPEITHI (Rammensee, H. G., et al.(1997) “MHC Ligands and Binding Motifs” Landes Bioscience, Georgetown),BIMAS (Parker, K. C., et al. (1994) J. Immunol. 152, 163-175),artificial neural networks (Bredenbeck, A., et al. (2005) J. Immunol.174, 6716-6724), and PePSSI (Bui, H. H., et al. (2006) Proteins 63,43-52); and experimental methods using peptide libraries (Rodda, S. J.(2002) J. Immunological Methods 267, 71-77; Sospedra, M., et al. (2003)Methods 29, 236-247; Pinilla, C., et al. (2001) Cancer Res. 61,5153-5160), and mass spectroscopy (Lemmel, C. and Stevanovic, S. (2003)Methods 29, 248-259; Lawendowski, C. A., et al. (2002) J. Immunol. 169,2414-2421).

In some embodiments, the present invention involves directly measuringthe affinity of the MHCI-peptide interaction using a library ofcandidate peptides and selecting from the library those peptides thatshow intermediate to moderately weak binding affinity for the MHCI ofinterest. One widely used method for measuring the binding affinity ofpeptides to MHCI is to perform a competition-based peptide-binding assayin which the inhibition of binding of a fixed concentration of aradiolabelled standard peptide is measured in the presence of varyingconcentrations of a test peptide (Ruppert, J., et al. (1993) Cell 74,929-937; Sette, A., et al. (1994) J. Immunol. 153, 5586-5592; Bullock,T. N. J., et al. (2000) J. Immunol. 164, 2354-2361). In someembodiments, the relative affinity of a peptide binding to MHCI can bemeasured using cells deficient in TAP (transporter associated withantigen processing) which leads to expression on the cell surface ofMHCI complexes without a bound peptide (Rock, K. L. and Goldberg, A. L.(1999) Annu. Rev. Immunol. 17, 730-779). As in the previous assay,binding affinity is measured relative to a standard peptide (Gross,D-A., et al. (2004) J. Clin. Invest. 113, 425-433).

Using the competition-based peptide-binding assay, candidate parentpeptides have been classified into four categories: good binders with aK_(D)≤50 nM, intermediate binders with a K_(D) of 50 to 500 nM, weakbinders with a K_(D) of 500 nM to 50 uM, and non-binders with a K_(D)>50uM (Ruppert, J., et al. (1993) Cell 74, 929-937; Sette, A., et al.(1994) J. Immunol. 153, 5586-5592). Further studies have been performedin which peptide binding affinity has been correlated withimmunogenicity tested by measuring cytotoxic T cell responses. In thesestudies, all peptides that bind to MHCI with high affinity (K_(D)≤50 nM)are immunogenic, only some peptides that bind with intermediate affinity(50 to 500 nM) are immunogenic, and none of the peptides that bind withweak affinity (500 nM to 50 uM) are immunogenic (Sette, A., et al.(1994) J. Immunol. 153, 5586-5592; van der Burg, S. H., et al. (1996) J.Immunol. 156, 3308-3314). Exceptions to these correlations have beenidentified, but they appear to occur only rarely. These categories,based on peptide binding affinities, can be used to guide the selectionof preferred candidate parent peptides where the preferred candidateswould be those that bind to MHCI with intermediate to moderately weakaffinity. Peptides with high binding affinity (good binders) most likelywould not be selected since it is more likely that these self-peptideswould not be immunogenic in patients due to selective elimination of Tcells by thymic education and/or T cell tolerance. Parent peptides thatbind with weak affinity would also not be preferred since there is ahigher likelihood that these peptides would not be displayed on thesurface of target cells. In addition, the stability of the MHCI-peptidecomplex has also been found to affect the immunogenicity of peptides(van der Burg, S. H., et al. (1996) J. Immunol. 156, 3308-3314).Consequently, measurements of MHCI-peptide complex stability are alsoimportant in the selection of preferred candidate parent peptides withcomplex half-lives greater than 3 hours correlating better withimmunogenicity.

To implement this method, in some embodiments, a TAA or other targetantigen is selected from which to identify one or more peptides ofpotential interest, and such peptides are used to prepare one or morepotential derivative peptides for testing. The target antigen can beobtained from any cancer cell or pathogenic organism or infected cellfrom which a peptide can be selected for use in preparing a peptidederivative of the present invention useful to treat or prevent acondition or disorder in a subject in need of such treatment orprevention.

A candidate parent peptide may also be selected from a library. Alibrary of peptides of 8, 9, 10 or 11 contiguous residues of the targetantigen is constructed by any convenient method known in the art suchas, e.g., by routine solid phase synthesis methods. The library may beof uniform size or mixed. Preferably the library is a “complete library”consisting of all possible such peptides of a specific size. The size ofa library depends upon the length of the target protein and the lengthof the library's peptides. For example, a complete library of 10-mers ofa 250-residue target will contain 240 peptides. A representative libraryconsisting of at least a tenth of the members of the complete librarycan also be used. The library may be “edited” to exclude certainpeptides such as those that might cause chemical instability (e.g.,those containing cysteines and methionines), or that would bepractically insoluble.

After the library is constructed, peptides having intermediate tomoderately weak affinity for MHCI are identified as candidate parentpeptides for further analysis. Parent peptides will preferably have aMHCI binding affinity value above 50 nM, but preferably below 50 uM asdiscussed above. Different screening methods can be used including thecompetition-based peptide-binding assay and the cell-based binding assayusing TAP deficient cells. However, if the binding affinity ranges areused to select peptides, it will be important to use the same peptidebinding assay or to run sufficient controls to correlate the resultsfrom the alternative assay with the competition-based peptide-bindingassay cited herein.

Candidate parent peptides may also be known peptides chosen on the basisof published binding data or on the basis of analysis using analgorithm, or both, as noted above.

Once a peptide is identified as a candidate parent peptide formodification, a modified peptide can be constructed by substituting anamino acid residue at an anchor position in the peptide with anon-natural amino acid (e.g., conformation constraining non-naturalamino acid), and the modified peptide tested for extent ofcross-reactivity by the activated T-cells on the desired target cell. Ifa wild type residue at an anchor position is the most highly conservedresidue, then the replacement may be selected from among non-naturalamino acids chemically similar to the most highly conserved residue. Ifthe wild type residue at the anchor position is not the most highlyconserved residue, then it may be replaced with non-natural amino acidschemically similar to the most highly conserved residue. For example, aparent peptide may be a HLA-A2 peptide with an amino acid other than Leuat P2. A peptide derivative of this parent peptide may be a peptide thatcontains a non-natural peptide that is chemically similar to Leu, suchas α-methyl leucine at P2. A peptide derivative prepared according tothe method of, and suitable for practicing the present inventionpreferably displays an affinity value of less than 500 nM, and morepreferably less than 50 nM to increase the likelihood that the peptidederivative will be immunogenic and elicit an immune response in patientswhen used as a component in a vaccine.

Screening for evaluation of MHCI-peptide affinity or MHCI-peptidecomplex stability can be performed by any known method. Such methods canbe divided into cell-free methods, e.g., using isolated,recombinantly-produced MEW products (see section 5.1 in the Examples;Sette. A. et al. (1994) Mol. Immunol. 31:813-22; and Chen, Y. et al.(1994) J. Immunol. 152:2874-81), and cell-based methods (e.g., usingMHCI produced by cells lacking a functional peptide transporter enzyme,which therefore do not form MHCI complexes with endogenous peptides).Valmori, D., et al., J. Immunol. 161, 6956-6962 (1998); Sarobe, et al.,J. Clin. Invest. 102, 1239-1248 (1998); Gross, D-A., et al., J. ClinInvest. 113, 425-433 (2004). See section 5.1 in the Examples.

A method for measuring peptide binding affinity to isolated MHCI isreported in Sette, A., 1994, Mol. Immunology 31, 813-22. Those skilledin the art will appreciate that the calculated affinity based onmeasured on-rates and off-rates is dependent, to some extent, ontechnical factors, with temperature as one of the most sensitive.Because peptide-free MHCI products are unstable at 37° C., bindingaffinity is conventionally measured at 23° C. Hereafter, when stated asa limitation, binding affinity in a cell free assay will refer to thenumerical value that would be obtained according to the method of Setteet al., at 23° C. Values determined by different assay methods and underdifferent conditions may not be directly comparable to values determinedusing the method of Sette et al., so controls must be used that allowcomparisons on relative terms, i.e., a given derivative is more or lesspotent than a given control.

Cell-based screening assays utilizing a cell line lacking a functionalpeptide transporter enzyme can be divided into two types: (i) affinityassays, and (ii) stability assays. In an example of an affinity assay,TAP-deficient cells are incubated overnight with a particular testpeptide, washed, and the amount of the relevant MHCI-peptide complexfound on their surface analyzed as a function of peptide concentration.Sarobe et al., J. Clin. Invest. 102:1239-48 (1998); and Gross et al., J.Clin. Invest. 113:425-33 (2004). The concentration required to providesome fraction of maximum display is proportional to the binding affinityof the peptide to MHCI. The results are typically reported as relativeaffinity where the binding of the test peptide is compared to thebinding of a standard peptide that has been shown to typically bind withhigh affinity to MHCI (K_(D)≤50 nM (Ruppert, J., et al. (1993) Cell 74,929-937; Sette, A., et al. (1994) J. Immunol. 153, 5586-5592).

In an example of a stability assay, cells are incubated with or withoutexcess peptide overnight, the cells are washed free of unbound peptide,and then subjected to a pharmacologic blockade (e.g., with Brefeldin(Sigma-Aldrich)) of the expression of newly synthesized MHC. The amountof relevant cell-surface MHC can be measured at time points followinginstitution of the blockade. Results from stability assays are oftenreported as DC₅₀ (dissociation complex) values, which is the timerequired for the loss of 50% of the MHCI/peptide complexes that werepresent on the surface of the cells at t=0. Typically, the time to reachhalf maximum binding for high affinity peptides is greater than 8 hours.

The stability assay is considered a measure of the “off-rate” of thepeptide from the MHC-peptide complex, while affinity is affected by boththe “on-rate” and the “off-rate”. There are theoretical grounds forconsidering the “off-rate” the more important factor for determining theeffective activity of an antigen, which has had some empirical support.Vertuani, S., et al., 2004, J. Immunol. 172, 3501-8. For the presentinvention, this distinction is not critical. Screening can be performedeither by affinity or stability assays. Those skilled in the art willappreciate that technology specially adapted for the measurement ofMHC-peptide complex interactions can be employed with particularadvantage in the practice of the present invention. See U.S. Pat. No.5,635,363 and No. 5,723,584. Useful technology is also availablecommercially from Beckman Coulter, Inc. under the trademark “iTopia™”Epitope Discovery System.

After finding a parent peptide having suitable binding characteristics,the present invention requires at least one suitable modification to thepeptide so as to preferably increase immunogenicity. That modificationinvolves replacing at least one primary anchor residue for theparticular MHC of interest with an appropriate non-natural amino acid.In one embodiment, for HLA A2 epitopes, the primary anchor residue thatis substituted is at the P2 position in a 9-mer or a 10-mer. In anotherembodiment, there are two substitutions, where the first substitution isat P2 and the second substitution is at the terminal residue position(i.e., PΩ). For example, for a 9-mer, there is a substitution at both P2and P9; and for a 10-mer, there is a substitution at both P2 and P10.

Once a peptide has been modified by appropriate substitution, it can betested to determine if it is a peptide derivative of the presentinvention. As described above, a peptide derivative of the presentinvention triggers an expansion of T cells that recognize the parentpeptide. Preferably, the peptide derivative is also more immunogenicthan its parent peptide and preferably will exhibit at least one, atleast two, at least three, at least four, or all five of the followingproperties compared to the corresponding parent peptide: (a) itgenerates a T-cell immune response that is greater than the T-cellimmune response generated by the parent peptide; (b) it binds to MHCIwith an affinity that is higher than the affinity with which the parentpeptide binds to MHCI, i.e., the peptide derivative has a lower K_(D)than the parent peptide; (c) the affinity of T-cell receptors for thecomplex formed between MHCI and a peptide derivative of the presentinvention is higher than the affinity of T-cell receptors for thecomplex formed between MHCI and the parent peptide; (d) a complex formedbetween MHCI and a peptide derivative of the present invention is morestable (i.e., has a slower off-rate) than a complex formed between MHCIand the parent peptide; and (e) the peptide derivative of the presentinvention triggers an expansion of a broader number of T-cell clonesthat recognize the parent peptide than are triggered by the parentpeptide.

4.3. Synthesis of Peptide Derivatives

Peptide derivatives of the present invention can be prepared by anysuitable method, e.g., by solid phase synthesis. The coupling of aminoacids and amino acid analogs can be accomplished by techniques familiarto those in the art and described, e.g., in Stewart and Young, 1984,“Solid Phase Synthesis”, Second Edition, Pierce Chemical Co., Rockford,Ill. Amino acids and amino acid analogs used for peptide synthesis canbe standard Boc (N^(α)-amino-protected N^(α)-t-butyloxycarbonyl) aminoacid or amino acid analog resin with standard deprotecting,neutralization, coupling and wash protocols of the original solid phaseprocedure of Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154), or thebase-labile N^(α)-amino protected 9-fluorenylmethoxycarbonyl (Fmoc)amino acids first described in Carpino and Han (1972, J. Org. Chem.37:3403-3409). Both Fmoc and Boc α-amino protected amino acids and aminoacid analogs can be obtained from Fluka, Bachem, Advanced Chemtech,Sigma, Cambridge Research Biochemical, Peninsula Labs, or other chemicalcompanies familiar to those who practice in this field. In addition, theinvention can be practiced with other N^(α)-protecting groups familiarto those of skill in the art.

Many methods of activation can be used in the practice of the presentinvention and include, e.g., the use of preformed symmetrical anhydride(PSA), preformed mixed anhydride (PMA), acid chlorides, active esters,and in situ activation of the carboxylic acid, as described by Fieldsand Noble (1990, Int. J. Pept. Protein Res. 35:161-214).

Solid phase peptide synthesis can be accomplished by techniques familiarto those in the art in view of the present disclosure, and described,e.g., in Stewart and Young, supra, as well as in Fields and Noble,supra, or by using automated synthesizers such as sold by ABS.

The completeness of coupling should be assessed. Those skilled in theart will be familiar with well known quantitative monitoring tests suchas ninhydrin (the Kaiser test), picric acid,2,4,6-trinitro-benzenesulfonic (TNBS), fluorescamine, and chloranil,which are based on reagent reaction with free amino groups to produce achromophoric compound. If imino acids (e.g., Pro and Hyp) are used,isatin monitoring is a preferred method (Fields and Noble, supra).Quantification of reaction completeness can be monitored during thecourse of the reaction as described, e.g., by Salisbury et al.(International PCT Publication WO 91/03485). If the coupling reaction isincomplete as determined by this test, the reaction can be forced tocompletion by several methods familiar to those in the art, including:(a) a second coupling using a one- to five-fold excess of protectedamino acid or amino acid analog; (b) an additional coupling usingdifferent or additional solvents (e.g., trifluoroethane); or (c) theaddition of chaotropic salts, e.g., NaClO₄ or LiBr (Klis an Stewart,“Peptides: Chemistry, Structure and Biology”; in Rivier and Marshall(eds) ESCOM Publ. 1990, pp. 904-906).

Once synthesized, a peptide derivative of the present invention can becovalently or non-covalently complexed to a macromolecular carrier,including, but not limited to, natural and synthetic polymers, proteins,polysaccharides, poly(amino acid), polyvinyl alcohol, polyvinylpyrrolidone, and lipids. A peptide derivative of the present inventioncan be conjugated to a fatty acid for introduction into a liposome. U.S.Pat. No. 5,837,249. A peptide derivative of the present invention can becovalently or non-covalently complexed to a solid support. A peptidederivative of the present invention can be covalently bound to one ormore molecules of polyethylene glycol (PEG). A peptide derivative of thepresent invention can be associated with an antigen-presenting matrixwith or without co-stimulatory molecules, as known in the art.

4.4. Therapeutic Methods

The peptide derivative of the present invention may be administered aspart of a longer peptide or polypeptide and/or it may be fused to amoiety to increase its half life in vivo. The peptide derivative,polypeptide or fusion comprising said derivative, may be administered aspart of a pharmaceutically acceptable composition. The present inventionprovides a method of inducing an immune response by administering apeptide derivative of the invention to a subject, or by contacting acell(s) with the peptide derivative. The peptide derivative induces animmune response, e.g., against the corresponding parent peptide, targetantigen, tumor or cancer cell, or infectious agent.

In a preferred embodiment, the present invention provides a method oftreating or preventing a cancer. The method includes administering to asubject in need of such treatment a therapeutically effective amount ofan appropriate peptide derivative of the present invention. Effectivetreatment may require administration of multiple doses to maximize thetherapeutic effect.

Cancers, including any disease, disorder or symptom characterized byuncontrolled cell growth, in which cancer cells express acancer-associated antigen as described herein having immunogenicproperties relevant to human cancers, can be treated by administering toa subject in need of said treatment or prevention an appropriate peptidederivative of the present invention. Whether a particular therapeuticagent is effective in treating or preventing a certain type of cancercan be determined by any method known in the art.

Peptide derivatives of the present invention are useful as therapeuticagents to treat or prevent a cancer selected from, but not limited to,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendothelio-sarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, lymphoma, leukemia,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,hepatocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, cervical cancer, testicular tumor, lungcarcinoma, small cell lung carcinoma, bladder carcinoma, epithelialcarcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma.

In a preferred embodiment, peptide derivatives of the present inventionare useful as agents to treat or prevent melanoma. In said embodiment,the peptide derivatives are preferably MART-1-based peptide derivatives.

In a further preferred embodiment, peptide derivatives of the presentinvention are useful as agents to treat or prevent Survivin-expressingcancer. In said embodiment, the peptide derivatives are preferably basedon a peptide selected from the Survivin protein, in particular they areselected from the group consisting of the Survivin-based peptidederivatives of the present invention.

In certain embodiments of the present invention, the subject beingtreated with the peptide derivative of the present invention is alsotreated with one or more other therapeutic cancer treatments selectedfrom surgery, radiation therapy, immunotherapy, and chemotherapy. Inparticular, the therapeutic agent of the present invention used to treator prevent cancer can be administered in conjunction with one or morechemotherapeutic agents, such as, e.g., methotrexate, taxol, taxotere,mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide,ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin,dacarbazine, procarbizine, etoposides, campathecins, bleomycin,doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin,mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine,paclitaxel, docetaxel, among others. The peptide derivative and theother therapeutic treatment can be provided to a subject in any mannerand following any regimen that provides a benefit of combinationtherapy. Thus, the two treatments can be given simultaneously, orsequentially. The durations of treatment with the two therapies may bethe same or different.

Peptide derivatives of the present invention are also useful astherapeutic agents to treat or prevent infection including, but notlimited to, parasitic infections (such as those caused by plasmodialspecies, among others), viral infections (such as those caused byinfluenza viruses, leukemia viruses, immunodeficiency viruses such asHIV, papilloma viruses, herpes virus, hepatitis viruses, measles virus,poxviruses, mumps virus, cytomegalovirus, Epstein-Barr virus, amongothers), bacterial infections involving MHCI (such as those caused bystaphylococcus, streptococcus, pneumococcus, Neisseria gonorrhea,Borrelia, pseudomonas, mycobacteria, Salmonella, among others), oragainst any infectious agent that enters cells as part of its lifecycle.In certain embodiments of the present invention, the subject beingtreated with the peptide derivative of the present invention is alsotreated with one or more antibiotics or antivirals.

4.5. Pharmaceutical Formulations and Administration

The present invention further provides a pharmaceutical formulationcontaining a peptide derivative of the present invention combined with apharmaceutically acceptable carrier. In one embodiment, thepharmaceutical composition comprises a therapeutically effective amountof a peptide derivative of the present invention. Such formulations canbe varied depending upon the intended use and route of administration.

Many routes of administration can be selected to administer apharmaceutical composition of the present invention, including but notlimited to the oral, intracerebral, intradermal, intramuscular,intraperitoneal, intravenous, intra-arterial, subcutaneous, intranasal,vaginal, rectal, buccal, sublingual, transdermal, and mucosal, andscarification (i.e., scratching through the top layers of skin; see,e.g., Glenn et al., 1998, Nature 391:851; Glenn et al., 1998, J.Immunol. 161:3211-3214) routes, or by any other route of administrationdetermined to be appropriate under the circumstances of each case.

A suitable pharmaceutical composition can be formulated as aninjectable, either as a liquid solution or suspension. A solid formsuitable for solution or suspension in a liquid prior to injection mayalso be prepared. The formulation can also be emulsified. Thetherapeutic agent is often mixed with excipients that arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients include water, saline, buffered saline, dextrose,glycerol, ethanol, sterile isotonic aqueous buffer and the like, andcombinations thereof. In addition, the formulation may include minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents, and/or adjuvants that enhance the effectiveness ofthe vaccine. A peptide derivative of the invention can be introduced toa subject in microspheres or microcapsules, e.g., prepared from PLGA(see U.S. Pat. Nos. 5,814,344; 5,100,669; and 4,849,222; PCT PublicationNos. WO 95/11010 and WO 93/07861; Johansen et al., 2000, Vaccine 18:209-215). Alternatively, a peptide derivative of the invention can beencapsulated in liposomes (Ludewig et al., 2001, Vaccine 19:23-32;Copland et al., 2003, Vaccine 21: 883-890; Chang et al., 2001, Vaccine19:3608-3614). Alternatively, a peptide derivative of the invention canbe encapsulated in ISCOMs (Morein et al., 1984, Nature 308:457-460;Lenarczyk et al., 2004, Vaccine 22: 963-974). Alternatively, a peptidederivative of the invention can be encapsulated in virus like particles(VLPs) (Storni et al., 2004, J. Immunol. 172:1777-1785). Othertechnologies are available to enable alternative formulations.

The pharmaceutical composition of the present invention can be a liquidsolution, a suspension, an emulsion, a tablet, a pill, a capsule, asustained release formulation, a suppository, or a powder. For oraladministration, the formulation can be, e.g., a tablet or capsuleprepared by conventional means using pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); and wetting agents(e.g., sodium lauryl sulphate). The tablets can be coated by methodsknown in the art. Liquid preparations for oral administration can takethe form, e.g., of solutions, syrups, emulsions or suspensions, or theycan be presented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations can be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats), emulsifying agents (e.g., lecithin oracacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol or fractionated vegetable oils), and preservatives (e.g., methylor propyl-p-hydroxybenzoates or sorbic acid). The preparations can alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate.

Suitable pharmaceutical excipients are described in “Remington'sPharmaceutical Sciences” by E. W. Martin, 18^(th) Edition.

The components of the pharmaceutical composition can be supplied eitherseparately or mixed together in a unit dosage form, e.g., as a drylyophilized powder or water-free concentrate in a sealed container suchas a vial or sachette indicating the quantity of therapeutically activeagent. Where the composition is administered by injection, an ampoule ofsterile diluent can be provided so that the ingredients can be mixedprior to administration.

In a specific embodiment, lyophilized peptide derivative of the presentinvention is provided in a first container, and a second containercomprises a diluent, preferably a diluent consisting of an aqueoussolution of 50% glycerin, 0.25% phenol, and an antiseptic (e.g., 0.005%brilliant green).

The pharmaceutical composition may further comprise an adjuvantcomponent to assist in potentiating the response to the peptidederivative. The term “adjuvant” refers to a compound or mixture that maybe non-immunogenic when administered to a subject alone, but thataugments the subject's immune response to another antigen whenadministered conjointly with that antigen. The adjuvant can beadministered as part of a pharmaceutical composition of the presentinvention, or as a separate formulation. Adjuvants include, but are notlimited to, oil-emulsion and emulsifier-based adjuvants such as completeFreund's adjuvant, incomplete Freund's adjuvant, MF59, or SAF; mineralgels such as aluminum hydroxide (alum), aluminum phosphate or calciumphosphate; microbially-derived adjuvants such as cholera toxin (CT),pertussis toxin, Escherichia coli heat-labile toxin (LT), mutant toxins(e.g., LTK63 or LTR72), Bacille Calmette-Guerin (BCG), Corynebacteriumparvum, DNA CpG motifs, muramyl dipeptide, or monophosphoryl lipid A;particulate adjuvants such as immunostimulatory complexes (ISCOMs),liposomes, biodegradable microspheres, or saponins (e.g., QS-21);cytokines such as IFN-gamma, IL-2, IL-12 or GM-CSF; synthetic adjuvantssuch as nonionic block copolymers, muramyl peptide analogues (e.g.,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine,N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy]-ethylamine),polyphosphazenes, or synthetic polynucleotides, and surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,hydrocarbon emulsions, keyhole limpet hemocyanins (KLH), or imiquimod(Testerman et al., 1995, J. Leukocyte Biol. 58:537-545). Preferably,these adjuvants are pharmaceutically acceptable for use in humans.

Administration of a pharmaceutical composition of the present inventionto a subject is intended to treat or prevent a disease, disorder orsymptom by triggering the development of protective immunity in thesubject. Within the meaning of the present invention, protectiveimmunity may be partial or complete. In a specific embodiment of thepresent invention, protective immunity can be reflected by anyimprovement in any condition or symptom being treated, including any oneof the following: a slowing of disease progression, increasing length torelapse, decreased rate of tumor growth, tumor regression, decreasedmortality, etc.

The present invention further provides a pharmaceutical kit comprising acontainer comprising a therapeutically effective amount of a peptidederivative of the present invention. The kit may further comprise asecond container comprising a sterile diluent that can be used toreconstitute or dilute the peptide derivative in the first container tothe appropriate concentration for administration. The kit may furthercomprise a printed notice in a form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts which notice describes the use of the peptide derivative totreat or prevent a disease or disorder, and reflects approval by theagency of the manufacture, use or sale of the peptide derivative forhuman administration. The kit preferably contains one or more unitdosage forms of the therapeutic agent. The kit may comprise metal orplastic foil, such as a blister pack.

According to the therapeutic methods of the present invention, thepharmaceutical compositions described herein can be administered to asubject at therapeutically effective or immunogenically effective dose,and preferably, with minimal toxicity.

Following methodologies which are well-established in the art (see,e.g., reports on evaluation of several vaccine formulations in acollaborative effort between the Center for Biological Evaluation andFood and Drug Administration and the National Institute of Allergy andInfectious Diseases (Goldenthal et al., National Cooperative VaccineDevelopment Working Group, AIDS Res. Hum. Retroviruses, 1993,9:545-549)), effective doses and toxicity of compounds and compositionsof the instant invention can first be determined in preclinical studiesusing small animal models (e.g., mice) in which these compounds andcompositions have been found to be immunogenic and that can bereproducibly immunized by the same route proposed for the human clinicaltrials. Preferably, mice that are transgenic for the relevant human MHCImolecule are used.

In a specific embodiment, the efficiency of epitope-specific CD8+ T-cellresponses to the pharmaceutical and vaccine compositions of the presentinvention is determined by the enzyme-linked immunospot technique(ELISPOT). ELISPOT is a standard method in the art (Miyahira et al.,1995, J. Immunol. Meth., 181: 45-54; Guelly et al., 2002, Eur. J.Immunol., 32:182-192; Nikitina and Gabrilovich, 2001, Int. J. Cancer,94:825-833; Field et al., 2001, Immunol. Rev., 182:99-112; Altfeld etal., 2001, J. Immunol., 167:2743-2752; Skoberne et al., 2001, J.Immunol., 167:2209-2218). This method employs pairs of antibodiesdirected against distinct epitopes of a cytokine, and allows thevisualization of cytokine secretion by individual T-cells following invitro stimulation with an antigen. ELISPOT has the advantage ofdetecting only activated/memory T-cells, and the cytokine release can bedetected at single cell levels, allowing direct determination of T-cellfrequencies (Czerkinsky et al., 1988, J. Immunol. Methods, 25:29;Taguchi et al., 1990, J. Immunol. Methods, 128:65). The cytokinecaptured by the immobilized antibody in the ELISPOT assay can bedetected in situ using an insoluble peroxidase substrate. Thus, thecytokine secretion by individual cells can be clearly visualized. Thehigh sensitivity and easy performance, allowing a direct enumeration ofpeptide-reactive T-cells without prior in vitro expansion, make theELISPOT assay well suited to monitor and measure T-cell responses,particularly CD8+ T-cell responses of very low frequencies. According toalternative embodiments, the efficiency of an epitope-specific CD8+T-cell response to a peptide derivative or pharmaceutical composition ofthe present invention can be determined using other art-recognizedimmunodetection methods such as, e.g., ELISA (Tanguay and Killion, 1994,Lymphokine Cytokine Res. 13:259) and intracellular staining (Carter, L.L. and Swain, S. L. 1997, Curr. Opin. Immunol. 9, 177-182).

For any peptide derivative or pharmaceutical composition used in atherapeutic method of the present invention, the therapeuticallyeffective dose can be estimated initially from animal models and basedon knowledge in the art to achieve a dose that induces an immuneresponse. In safety determinations for each composition, the dose andfrequency of immunization should preferably meet or exceed thoseanticipated for use in the clinical trial.

The choice and amount of peptide derivative, and the selection andamount of other components in the pharmaceutical composition, depend onthe disease, disorder or symptom being treated, as well as on the routeof administration. Determination of the proper dose and treatmentregimen will typically vary depending on the circumstances andconditions of the particular subject being treated, including age, bodyweight, gender, general health conditions, sensitivity, currentadministration of other drugs, and stage or seriousness of the disease,disorder or symptom being treated. The appropriate dose and dosage timesshould preferably be decided according to the judgment of a medicalpractitioner and each subject's circumstances according to standardclinical techniques and in view of published clinical reports. Theappropriate pharmaceutical composition can be administered continuouslyor intermittently depending on a subject's response to treatment, andeither alone or in combination with one or more other therapeutic agentsto treat the same or a different disease, disorder, condition orsymptom. Effective doses may be extrapolated from dose-response curvesderived from animal model test systems, including transgenic animalmodels.

The following examples further describe the materials and methods usedin carrying out the invention. The examples are not meant to limit theinvention in any manner.

5. EXAMPLES 5.1. Affinity and Stability of HLA-A201/Peptide Complexes

Modified peptides were tested for their ability to bind to and stabilizethe HLA-A0201 molecule using one of two assays: (i) a live cell/flowcytometry-based assay; or (ii) a soluble class I/plate based-assay.E.g., Sharma et al., J. Biol. Chem. 276:21443-49 (2001); Pogue et al.,PNAS 92:8166-70 (1995).

I. Live Cell/Flow Cytometry-Based Assays

Relative Binding Affinity.

The live cell/flow cytometry-based assay is a well-established assayutilizing the TAP-deficient hybridoma cell line T2 (American TypeCulture Collection (ATCC Accession No. CRL-1992), Manassas, Va.). TheTAP deficiency in this cell line leads to inefficient loading of MHCI inthe ER and an excess of empty MHCIs. Salter and Cresswell, EMBO J.5:943-49 (1986); Salter, Immunogenetics 21:235-46 (1985). Empty MHCIsare highly unstable, and are therefore short-lived. When T2 cells arecultured at reduced temperatures, empty MHCIs appear transiently on thecell surface, where they can be stabilized by the exogenous addition ofMHCI-binding peptides. To perform this binding assay, peptide-receptiveMHCIs were induced by culturing aliquots of 10⁷ T2 cells overnight at26° C. in serum free AIM-V medium alone, or in medium containingescalating concentrations (0.1 to 100 uM) of peptide. Cells were thenwashed twice with PBS, and subsequently incubated with a fluorescenttagged HLA-A0201-specific monoclonal antibody, BB7.2, to quantify cellsurface expression. Samples were acquired on a FACS Calibur instrument(Becton Dickinson) and the mean fluorescence intensity (MFI) determinedusing the accompanying Cellquest software.

The specific increase in mean fluorescent intensity (MFI) was used as ameasure of peptide binding, and was calculated as follows:

$\frac{{MFI}\mspace{14mu}{with}\mspace{14mu}{modified}\mspace{14mu}{peptide}}{{MFI}\mspace{14mu}{without}\mspace{14mu}{peptide}} \times 100$The relative efficiency with which a peptide stabilizes an MHCI at thesurface of these cells is used as a measure of relative bindingaffinity. To determine the relative binding affinity, maximal bindingwas determined using the influenza matrix₅₈₋₆₆ peptide as a control,which peptide has been shown to bind to HLA-A0201 with high affinity.The relative binding affinity for each test peptide was taken as theconcentration of the test peptide at which 50% maximal binding occurs.

Assessment of Peptide/HLA-A0201 Complex Stability.

Aliquots of 10⁷ T2 cells were cultured overnight at 26° C. in serum-freeAIM-V medium alone, or in medium containing peptide at a concentrationof 100 uM. Cells were then incubated with Brefeldin A at 10 ug/ml for 1hour, washed, and subsequently incubated at 37° C. for 0, 2, 4 or 6hours in the presence of 0.5 ug/ml Brefeldin A. Cells were washed andstained with fluorescently tagged BB7.2 to quantify cell surfaceexpression of HLA-A0201. The time required for the loss of 50% of thecomplexes stabilized at time 0 (DC₅₀) was used as a measure of thestability of the peptide/HLA-A0201 complex.

II. Soluble Class I/Plate-Based Assays

Relative Binding Affinity.

The plate-based assay, named iTopia™, is a commercial assay (BeckmanCoulter). The assay is performed in wells of microtiter plates. At theoutset of the assay, MHCI complexes (properly folded MHC heavy chain,beta 2 microglobulin and a place-holder peptide), are tethered tostreptavidin-coated microplate wells. (Proper formation/folding of theMHC complex may be determined by binding of an anti-HLA-ABC antibody.) Abuffer designed to unfold and dissociate the complex was added to eachtest well, allowed to incubate, and the place holder peptide and beta 2microglobulin were washed away. Escalating concentrations of the testpeptide, along with additional beta 2 microglobulin, were added to eachwell and incubated in a buffer designed to promote refolding ofcomplexes. Plates were incubated overnight at 21° C. Properly foldedMHCI complex molecules were detected with a fluorescently labeledantibody selected based on its ability to only bind properly folded MHCIcomplexes. Binding was reported relative to a standardized positivecontrol peptide. Relative affinities were reported as ED₅₀, and aredefined herein as the concentration of peptide required to achieve halfmaximal binding of the positive control. The lower the ED₅₀ value, thestronger the binding of the peptide to MHCI.

Assessment of Peptide/HLA-A0201 Complex Stability.

MHCI complexes were dissociated in each test well using strippingbuffer, and the place-holder peptide and beta 2 microglobulin werewashed away. 100 uM of the test peptide was added to appropriate wellsalong with additional beta 2 microglobulin and the anti-HLA ABCantibody, and the plates were incubated overnight at 21° C. Plates werewashed, and fresh buffer was added to each well. Plates were shifted to37° C., and the appropriate wells were read at 0, 0.5, 1, 1.5, 2, 4, 6and 8 hours. The half-life of the complex was used as a measure of itsstability, and is calculated as the amount of time required for loss of50% of the complexes formed at time 0.

5.2. Immunogenicity of Peptides

Generation of Peptide Specific T-Cells.

In vitro education (IVE) assays were used to test the ability of eachtest peptide to expand CD8⁺ T-cells. Mature professional APCs wereprepared for these assays in the following way. 80-90×10⁶ PBMCs isolatedfrom a healthy human donor were plated in 20 ml of RPMI media containing2% human AB serum, and incubated at 37° C. for 2 hours to allow forplastic adherence by monocytes. Non-adherent cells were removed and theadherent cells were cultured in RPMI, 2% human AB serum, 800 IU/ml ofGM-CSF and 500 IU/ml of IL-4. After 6 days, TNF-alpha was added to afinal concentration of 10 ng/ml. On day 7, the dendritic cells (DC) werematured either by the addition of 12.5 ug/ml poly I:C or 0.3 ug/ml ofCD40L. The mature dendritic cells (mDC) were harvested on day 8, washed,and either used directly or cryopreserved for future use.

For the IVE of CD8+ T-cells, aliquots of 2×10⁵ mDCs were pulsed witheach peptide at a final concentration of 100 uM, incubated for 4 hoursat 37° C., and then irradiated (2500 rads). The peptide-pulsed mDCs werewashed twice in RPMI containing 2% human AB serum. 2×10⁵ mDCs and 2×10⁶autologous CD8⁺ cells were plated per well of a 24-well plate in 2 ml ofRPMI containing 2% human AB, 20 ng/ml IL-7 and 100 pg/ml of IL-12, andincubated for 12 days. The CD8⁺ T-cells were then re-stimulated withpeptide-pulsed, irradiated mDCs. Two to three days later, 20 IU/ml IL-2and 20 ng/IL7 were added. Expanding CD8⁺ T-cells were re-stimulatedevery 8-10 days, and were maintained in media containing IL-2 and IL-7.Cultures were monitored for peptide-specific T-cells using a combinationof functional assays and/or tetramer staining. Parallel IVEs with themodified and parent peptides allowed for comparisons of the relativeefficiency with which the peptides expanded peptide-specific T-cells.

5.3. Quantitative and Functional Assessment of CD8⁺ T-Cells

Tetramer Staining.

MHC tetramers were purchased from Beckman Coulter (San Diego, Calif.),and were used to measure peptide-specific T-cell expansion in the IVEassays. For the assessment, tetramer was added to 1×10⁵ cells in PBScontaining 1% FCS and 0.1% sodium azide (FACS buffer) according tomanufacturer's instructions. Cells were incubated in the dark for 20 minat room temperature. Antibodies specific for T-cell markers, such asCD8, were then added to a final concentration suggested by themanufacturer, and the cells were incubated in the dark at 4° C. for 20min. Cells were washed with cold FACS buffer and resuspended in buffercontaining 1% formaldehyde. Cells were acquired on a FACS Calibur(Becton Dickinson) instrument, and were analyzed by use of Cellquestsoftware (Becton Dickinson). For analysis of tetramer positive cells,the lymphocyte gate was taken from the forward and side-scatter plots.Data were reported as the percentage of cells that were CD8⁺/Tetramer⁻.

ELISPOT.

Peptide-specific T-cells were functionally enumerated using the ELISPOTassay (BD Biosciences), which measures the release of IFNgamma fromT-cells on a single cell basis. Target cells (T2 or HLA-A0201transfected C1Rs) were pulsed with 10 uM peptide for 1 hour at 37° C.,and washed three times. 1×10⁵ peptide-pulsed targets were co-cultured inthe ELISPOT plate wells with varying concentrations of T-cells (5×10² to2×10³) taken from the IVE culture. Plates were developed according tothe manufacturer's protocol, and analyzed on an ELISPOT reader (CellularTechnology Ltd.) with accompanying software. Spots corresponding to thenumber of IFNgamma-producing T-cells were reported as the absolutenumber of spots per number of T-cells plated. T-cells expanded onmodified peptides were tested not only for their ability to recognizetargets pulsed with the modified peptide, but also for their ability torecognize targets pulsed with the parent peptide.

CD107 Staining.

CD107a and b are expressed on the cell surface of CD8⁺ T-cells followingactivation with cognate peptide. The lytic granules of T-cells have alipid bilayer that contains lysosomal-associated membrane glycoproteins(“LAMPs”), which include the molecules CD107a and b. When cytotoxicT-cells are activated through the T-cell receptor, the membranes ofthese lytic granules mobilize and fuse with the plasma membrane of theT-cell. The granule contents are released, and this leads to the deathof the target cell. As the granule membrane fuses with the plasmamembrane, C107a and b are exposed on the cell surface, and therefore aremarkers of degranulation. Because degranulation as measured by CD107 aand b staining is reported on a single cell basis, the assay is used tofunctionally enumerate peptide-specific T-cells. To perform the assay,peptide was added to HLA-A0201-transfected cells C1R to a finalconcentration of 20 uM, the cells were incubated for 1 hour at 37° C.,and washed three times. 1×10⁵ of the peptide-pulsed C1R cells werealiquoted into tubes, and antibodies specific for CD107 a and b wereadded to a final concentration suggested by the manufacturer (BectonDickinson). Antibodies are added prior to the addition of T-cells inorder to “capture” the CD107 molecules as they transiently appear on thesurface during the course of the assay. 1×10⁵ T-cells from the IVEculture were added next, and the samples were incubated for 4 hours at37° C. The T-cells were further stained for additional cell surfacemolecules such as CD8 and acquired on a FACS Calibur instrument (BectonDickinson). Data was analyzed using the accompanying Cellquest software,and results were reported as the percentage of CD8⁺ CD107 a and b⁺cells.

CTL Lysis.

Cytotoxic activity was measured using a chromium release assay. TargetT2 cells were labeled for 1 hour at 37° C. with Na⁵¹Cr and washed. 5×10³target T2 cells were then added to varying numbers of T-cells from theIVE culture. Chromium release was measured in supernatant harvestedafter 4 hours of incubation at 37° C. The percentage of specific lysiswas calculated as:

$\frac{{{Experimental}\mspace{14mu}{release}} - {{spontaneous}\mspace{14mu}{release}}}{{{Total}\mspace{14mu}{release}} - {{spontaneous}\mspace{14mu}{release}}} \times 100$

5.4. Results

Table 2. Using the iTopia™ plate-based assay system from BeckmanCoulter, the stability of complexes formed between the MART-1 peptidederivatives and HLA-A2 were determined. Several of the peptidederivatives formed complexes with HLA-A2 that were significantly morestable than those formed with the native MART-1 peptide (SEQ ID NO: 1).Derivative peptides with SEQ ID NOs: 7 and 16 formed complexes withHLA-A2 that had half-lives greater than 100 hours.

Table 3. An ELISPOT assay measuring IFNgamma production (a marker ofCD8+ T-cell activation) was used to determine if CD8+ T-cells expandedin the presence of dendritic cells pulsed with the MART-1 peptidederivatives would recognize target cells pulsed with the native MART-1peptide. For three of the four peptide derivatives tested (SEQ ID NOs:15, 16 and 18), the expanded T-cells recognized equally well targetcells pulsed with either the native MART-1 peptide or the peptidederivatives as indicated by the similarly high levels of IFNgammaproduced.

Table 4. CD107 a and b are markers of CD8+ T-cell activation anddegranulation. Analyzing CD107 expression using a FACS Caliburinstrument, CD8+ T-cells expanded in the presence of dendritic cellspulsed with the MART-1 peptide derivatives were tested for their abilityto recognize target cells pulsed with the native MART-1 peptide. Allfour of the CD8+ T-cell populations expanded with the derivativepeptides, recognized target cells pulsed with the native MART-1 peptideas shown by the increased levels of CD107 expression detected.

Table 5. The stability of complexes formed using HLA-A2 with nativesurvivin peptides and survivin peptide derivatives was tested using theiTopia™ plate-based assay system from Beckman Coulter. For both examplestested, the peptide derivative was found to form a more stable complexwith HLA-A2 than the corresponding native peptide (compare SEQ ID NO: 21to 26 and SEQ ID NO: 31 to 36). Notably, the survivin peptide derivative(SEQ ID NO: 36) showed a greater than 20-fold increase in the half lifeof the HLA-A2 peptide complex when compared to the native survivinpeptide.

TABLE 2 Stability of HLA-A2/MART-1 Peptide Derivative ComplexPeptide Used In SEQ ID Half Life of Binding Assay NO: Complex (hr)AAGIGILTV¹  1    1.4 A-[cpg]-GIGILTV  7 >100 E-[c3a]-AGIGILTV 13   23.6E-[chg]-AGIGILTV 15   31.0 E-[cpg]-AGIGILTV 16 >100 E-[dfb]-AGIGILTV 17  13.9 E-[dhl]-AGIGILTV 18    5.5 E-[phg]-AGIGILTV 19    6.5E-[sta]-AGIGILTV 20   15.3 E-[c5g]-AGIG-[amv]-LTV 49   11.9 ¹NativeMART-1 nonamer peptide

TABLE 3 T-Cells Expanded on MART-1 Peptide DerivativeRecognize Targets Pulsed with the Native MART-1 Peptide IFNγ Pro- IFNγPro- duction  duction  for Targets for Targets  Pulsed with SEQPulsed with MART-1 Peptide Used to ID Native Peptide Generate T-CellsNO: MART-1 Derivative AAGIGILTV  1  11 E-[chg]-AGIGILTV 15 279 261E-[cpg]-AGIGILTV 16 182 273 E-[dhl]-AGIGILTV 18 336 239 E-[phg]-AGIGILTV19  35  30

TABLE 4 T-Cells Expanded on MART-1 Peptide DerivativesRecognize Targets Pulsed with the Native MART-1 Peptide %CD8⁺ T-cells%CD8⁺ T-cells Degranulating Degranulating (CD107⁺) for Targets(CD107⁺) for Targets Pulsed Peptide Used to Sequence Pulsed with Nativewith MART-1 Peptide Generate T-Cells ID No. MART-1 Derivative AAGIGILTV 1  4 E-[chg]-AGIGILTV 15 56 61 E-[cpg]-AGIGILTV 16 37 60E-[phg]-AGIGILTV 19 11 20 E-[sta]-AGIGILTV 20 32 37

TABLE 5 Stability of Survivin Peptide/HLA-A2 Complexes Peptide Used inSequence Half Life of Binding Assay ID. No. Complex (hr) ISTFKNWPF² 21 0.24 I-[cpg]-TFKNWPF 26  1.9 KVRRAIEQL³ 31  1.9 K-[cpg]-RRAIEQL 36 50.6²Native Survivin 19-27 ³Native Survivin 130-138

While the invention has been described and illustrated with reference tocertain preferred embodiments thereof, those skilled in the art willappreciate that obvious modifications can be made herein withoutdeparting from the spirit and scope of the invention. Such variationsare contemplated to be within the scope of the appended claims.

All references cited herein are hereby incorporated by reference intheir entireties.

TABLE 6 HLA Class I Alleles Sero- Geno- Total No. NCBI type type AllelesEntry Reference A02 20101 89 AJ555412 Koller et al., J Immunology 134:2727-2733, 1988 A01 10101 11 AJ278305 Girdlestone, Nucleic Acid Research18: 6701-6701, 1990 A03 10101 18 X00492 Strachan et al., EMBO J 3:887-894, 1984 A11 101 20 X12781 Cowan et al., Immunogenetics 25:241-250, 1987 A24 20101 50 L47206 Magor et al., J Immunology 158:5242-5250, 1997 A68 101 30 AJ315642 Holmes et al., EMBO J 4: 2849-2854,1985 B07 201 40 M32317 Takiguchi et al., J Immunology 143: 1372-1378,1989 B08 1 22 M24036 Parham et al., PNAS USA 85: 4005-4009, 1988 B1510101 98 U03859 Hildebrand et al., Tissue Antigens 43: 209-218, 1994 B272 30 L38504 Seemann et al., EMBO J 5: 547-552, 1986 B35 101 56 M28115Ooba et al., Immunogenetics 30: 76-80, 1989 B40 101 63 U03698 Ways etal., Immunogenetics 25: 323-328 1987 B44 20101 43 M24038 Parham et al.,PNAS USA 85: 4005-4009, 1988 B51 101 41 M28205 Pohla et al.,Immunogenetics 29: 297-307, 1989 Cw03 23 23 M84172 Zemmour et al.,Tissue Antigens 39: 249-257, 1992 Cw04 17 17 M26432 Ellis et al., JImmunology 142: 3281-3285, 1989 Cw05 11 11 AJ010748 Baurain et al.,Tissue Antigens 53: 510-512, 1999 Cw06 11 11 M28160 Mizuno et al.,Immunogenetics 29: 323-330, 1989 Cw07 34 34 D38526 Wang et al., HumanImmunology 45: 52-58, 1996 Cw12 19 19 M28172 Takiguchi et al., JImmunology 143: 1372-1378, 1989

TABLE 7 HLA Binding Motifs Genotype Motif Reference A*01 xx[DE]xxxxx[Y]SYFPEITHI A*0101 xx[DE]xxxxx[Y] Marsh2000 A*0201 x[L(M)]xxxxxx[V(L)]Marsh2000 A*0201 x[LM]xxxxxx[VL] SYFPEITHI A*0202 x[L]xxxxxx[L]Marsh2000 A*0202 x[L(A)]xxxxxx[LV] SYFPEITHI A*0204 x[L]xxxxxx[L]Marsh2000 A*0204 x[L]xxxxxx[L] SYFPEITHI A*0205 x[V(QL)]xxxxxx[L]Marsh2000 A*0205 xxxxxxxx[L] SYFPEITHI A*0206 x[V(Q)]xxxxxxx Marsh2000A*0206 x[V(Q)]xxxxxx[V(L)] SYFPEITHI A*0207 x[L][D]xxxxx[L] Marsh2000A*0207 x[L]xxxxxx[L] SYFPEITHI A*0214 x[QV]xxx[K]xx[VL] Luscher2001A*0214 x[VQ(L)]xxxxxx[L] Marsh2000 A*0214 x[VQL(A)]xxxxxx[L(VM)]SYFPEITHI A*0217 x[L]xxxxxx[L] SYFPEITHI A*03 x[LVM]xxxxxx[KYF]SYFPEITHI A*0301 x[LVM(IAST)]xxxxxx[KY(FR)] Marsh2000 A*1101 xxxxxxxx[K]Marsh2000 A*1101 xxxxxxxx[KR] SYFPEITHI A*24 x[Y(F)]xxxxxx[ILF]SYFPEITHI A*2402 x[YF]xxxxxx[FWIL] Marsh2000 A*2402 x[YF]xxxxxx[LFI]SYFPEITHI A*2501 xxxxxxxxx[W] Yusim2004 A*2601 x[VTIFL]xxxxxx[YF]Marsh2000 A*2601 x[VTILF]xxxxxx[YF] SYFPEITHI A*2602x[VTILF]xxxxxx[YFML] Marsh2000 A*2602 x[VTILF]xxxxxx[YF(ML)] SYFPEITHIA*2603 x[VFILT]xxxxxx[YFML] Marsh2000 A*2603 x[VTILF]xxxxxx[YFML]SYFPEITHI A*2902 x[E(M)]xxxxxx[Y(L)] Marsh2000 A*2902x[E(M)]xxxxxx[Y(L)] SYFPEITHI A*3001 x[YF(VLMIT)]xxxxxx[L(YFM)]SYFPEITHI A*3002 x[YFLV]xxxxxx[Y] SYFPEITHI A*3003 x[FYIVL]xxxxxx[Y]SYFPEITHI A*3004 xxxxxxxx[YML] SYFPEITHI A*3101 xxxxxxxx[R] Marsh2000A*3101 xxxxxxxx[R] SYFPEITHI A*3201 x[I]xxxxxxx[W] Yusim2004 A*3303xxxxxxxx[R] Marsh2000 A*3303 xxxxxxxx[R] SYFPEITHI A*6601x[TV(APLIC)]xxxxxx[RK] SYFPEITHI A*6801 x[VT]xxxxxx[RK] Marsh2000 A*6801x[VT]xxxxxx[RK] SYFPEITHI A*6802 x[TV]xxxxxx[VL] Yusim2004 A*6901x[VT(A)]xxxxxx[VL] Marsh2000 A*6901 x[VTA]xxxxxx[VL(MQ)] SYFPEITHI B*07x[P]xxxxxx[LF] SYFPEITHI B*0702 x[P]xxxxxx[L(F)] Marsh2000 B*0702x[P(V)]xxxxxx[L] SYFPEITHI B*0703 x[P]xxxxxxx Marsh2000 B*0703x[P(ND)]xxxxxx[L] SYFPEITHI B*0705 x[P]xxxxxxx Marsh2000 B*0705x[P]xxxxxx[L(F)] SYFPEITHI B*08 xx[K(R)]x[KR]xxx[L(FM)] SYFPEITHI B*0801xx[K(R)]x[K(RH)]xxxx Marsh2000 B*0801 xx[K(R)]xxxxxx SYFPEITHI B*0802xx[K(RY)]x[K(H)]xxxx Marsh2000 B*0802 xx[K(RY)]x[K(H)]xxxx SYFPEITHIB*14 x[RK]xx[RH]xxx[L] SYFPEITHI B*1402 x[R(K)]xx[R(H)]xxx[L] Marsh2000B*1501 x[Q(LMVP)]xxxxxxx[YF] Marsh2000 B*1501 x[QL(MVP)]xxxxxxx[FY]SYFPEITHI B*1502 xxxxxxxx[YF(M)] Marsh2000 B*1502 x[QLVP]xxxxxx[FYM]SYFPEITHI B*1503 x[QK]xxxxxx[YF] SYFPEITHI B*1508 x[P(A)]xxxxxx[YF]Marsh2000 B*1508 x[PA]xxxxxx[YF] SYFPEITHI B*1509 x[H]xxxxxx[L(F)]Marsh2000 B*1509 x[H]xxxxxx[LFM] SYFPEITHI B*1510 x[H]xxxxxx[L(F)]SYFPEITHI B*1512 x[Q(LM)]xxxxxx[YF] SYFPEITHI B*1513 xxxxxxxx[W]Marsh2000 B*1513 x[LIQVPM]xxxxxx[W] SYFPEITHI B*1516x[T(S)]xxxxxx[Y(IVFM)] Marsh2000 B*1516 x[ST(F)]xxxxxx[IVYF] SYFPEITHIB*1517 x[TS]xxxx[L]x[Y(F)] Marsh2000 B*1517 x[TS]xxxxxx[YFLI] SYFPEITHIB*1518 x[H]xxxxxx[Y(F)] SYFPEITHI B*18 x[E]xxxxxxx Marsh2000 B*27x[R]xxxxxxx SYFPEITHI B*2701 x[RQ]xxxxxx[Y] Marsh2000 B*2701x[RQ]xxxxxx[Y] SYFPEITHI B*2702 x[R]xxxxxx[FY(ILW)] Marsh2000 B*2702x[R]xxxxxx[FYILW] SYFPEITHI B*2703 x[R(M)]xxxxxxx Marsh2000 B*2703x[R]xxxxxx[YF(RMWL)] SYFPEITHI B*2704 x[R]xxxxxx[YLF] Marsh2000 B*2704x[R]xxxxxx[YLF] SYFPEITHI B*2705 x[R(K)]xxxxxxx Marsh2000 B*2705x[R]xxxxxx[LFYRHK(MI)] SYFPEITHI B*2706 x[R]xxxxxx[L] Marsh2000 B*2706x[R]xxxxxx[L] SYFPEITHI B*2707 x[R]xxxxxx[L] Marsh2000 B*2707x[R]xxxxxx[LF] SYFPEITHI B*2709 x[R]xxxxxx[LVFIM] Marsh2000 B*2710x[R]xxxxxx[YF] Marsh2000 B*35 x[P(AVYRD)]xxxxxx[YFMLI] SYFPEITHI B*3501x[P(AV)]xxxxxxx Marsh2000 B*3501 x[P(AVYRD)]xxxxxx[YFMLI] SYFPEITHIB*3503 x[P(A)]xxxxxx[ML(F)] Marsh2000 B*3503 x[P(MILFV)]xxxxxx[ML(F)]SYFPEITHI B*3505 x[P]xxxxxx[F] Kenneally2000 B*3701x[D(E)]xxxxx[FML][IL] Marsh2000 B*3701x[DE(HPGSL)]xxxxx[FML(QKYL)][IL(TENDQGH)] SYFPEITHI B*3801 xxxxxxxx[FL]Marsh2000 B*3801 xxxxxxxx[FL(I)] SYFPEITHI B*3901 x[RH]xxxxxx[L]Marsh2000 B*3901 x[RH]xxxxxx[L(VIM)] SYFPEITHI B*3902 x[KQ]xxxxxx[L]Marsh2000 B*3902 x[KQ]xxxxxx[L(FM)] SYFPEITHI B*3909 x[RH(P)]xxxxxx[LF]SYFPEITHI B*40 x[E]xxxxxx[LWMATR] SYFPEITHI B*4001 x[E]xxxxxx[L]Marsh2000 B*4001 x[E]xxxxxx[L] SYFPEITHI B*4002 x[E]xxxxxx[IAVL]Yusim2004 B*4006 x[E]xxxxxx[V] Marsh2000 B*4006 x[E(P)]xxxxxx[V(AP)]SYFPEITHI B*4201 x[P]xxxxxx[L] Yusim2004 B*44 x[E]xxxxxx[Y] SYFPEITHIB*4402 x[E]xxxxxx[YF] Marsh2000 B*4402 x[E(MILD)]xxxxxx[FY] SYFPEITHIB*4403 x[E]xxxxxx[YF] Marsh2000 B*4403 x[E(MILVD)]xxxxxx[YF] SYFPEITHIB*4601 xxxxxxxx[YF] Marsh2000 B*4601 x[M(I)]xxxxxx[YF] SYFPEITHI B*4801x[QK]xxxxxx[L] Marsh2000 B*4801 x[QK(M)]xxxxxx[L] SYFPEITHI B*5101xxxxxxxx[FI] Marsh2000 B*5101 x[APG(WF)]xxxxxx[VI(WMVL)] SYFPEITHIB*5102 x[APG]xxxxxx[IV] Marsh2000 B*5102 x[APG]xxxxxx[IV] SYFPEITHIB*5103 xxxxxxxx[VIF] Marsh2000 B*5103 x[APG(FW)]xxxxxx[VIF] SYFPEITHIB*5201 xxxxxxx[IV][IV] Marsh2000 B*5201 xxxxxxx[IV(MF)][IV(MF)]SYFPEITHI B*5301 x[P]xxxxxxx Marsh2000 B*5301 x[P]xxxxxx[WFL] SYFPEITHIB*5401 x[P]xxxxxxx Marsh2000 B*5401 x[P]xxxxxxx SYFPEITHI B*5501x[P]xxxxxxx Marsh2000 B*5501 x[P]xxxxxxx SYFPEITHI B*5502 x[P]xxxxxxxMarsh2000 B*5502 x[P]xxxxxxx SYFPEITHI B*5601 x[P]xxxxxxx Marsh2000B*5601 x[P]xxxxxx[A(L)] SYFPEITHI B*5701 x[ATS]xxxxxx[FW] Marsh2000B*5701 x[ATS]xxxxxx[FWY] SYFPEITHI B*5702 x[ATS]xxxxxx[FW] Marsh2000B*5702 x[ATS]xxxxxx[FW] SYFPEITHI B*5801 x[ATS]xxxxxx[WF] Marsh2000B*5801 x[AST(G)]xxxxxx[FW(Y)] SYFPEITHI B*5802 x[ST]xxx[R]xx[F]Marsh2000 B*5802 x[ST]xxx[R]xx[F] SYFPEITHI B*6701 x[P]xxxxxxx Marsh2000B*6701 x[P]xxxxxxx SYFPEITHI B*7301 x[R]xxxxxx[P] Marsh2000 B*7301x[R]xxxxxx[P] SYFPEITHI B*7801 x[PAG]xxxxxxx Marsh2000 B*7801x[PAG]xxxxx[A(KS)]x SYFPEITHI B*8101 x[P]xxxxxx[L] Yusim2004 Cw*0102xx[P]xxxxx[L] Marsh2000 Cw*0102 x[AL]xxxxxx[L] SYFPEITHI Cw*0103x[AL]xxxxxx[L] Yusim2004 Cw*0202 x[A]xxxxxx[L] Yusim2004 Cw*0203x[A]xxxxxx[L] Yusim2004 Cw*0301 xxxxxxxx[LFMI] SYFPEITHI Cw*0302x[A]xxxxxx[FWY] Yusim2004 Cw*0303 x[A]xxxxxx[LM] Yusim2004 Cw*0304x[A]xxxxxx[LM] Marsh2000 Cw*0304 x[A]xxxxxx[LM] SYFPEITHI Cw*0305x[A]xxxxxx[LM] Yusim2004 Cw*0306 x[A]xxxxxx[LM] Yusim2004 Cw*0307x[A]xxxxxx[LF] Yusim2004 Cw*0308 x[A]xxxxxx[LM] Yusim2004 Cw*0309x[A]xxxxxx[LM] Yusim2004 Cw*0401 x[YP]xxxxxxx Marsh2000 Cw*0401x[YPF]xxxxxx[LFM] SYFPEITHI Cw*0402 x[YP]xxxxxx[LF] Yusim2004 Cw*0403x[P]xxxxxx[LF] Yusim2004 Cw*0404 x[YP]xxxxxx[LF] Yusim2004 Cw*0405x[YP]xxxxxx[LF] Yusim2004 Cw*0406 x[P]xxxxxx[LF] Yusim2004 Cw*0501x[A]xxxxxx[LF] Yusim2004 Cw*0502 x[A]xxxxxx[LF] Yusim2004 Cw*0601xxxxxxxx[LIVY] SYFPEITHI Cw*0602 xxxxxxxx[L] Marsh2000 Cw*0602xxxxxxxx[LIVY] SYFPEITHI Cw*0603 x[ALP]xxxxxx[L] Yusim2004 Cw*0604x[RQ]xxxxxx[L] Yusim2004 Cw*0701 x[RHK]xxxxxx[Y] Yusim2004 Cw*0702xxxxxxxx[YFL] SYFPEITHI Cw*0703 x[YP]xxxxxx[YL] Yusim2004 Cw*0704x[RQ]xxxxxx[LM] Yusim2004 Cw*0705 x[RQ]xxxxxx[Y] Yusim2004 Cw*0706x[RHK]xxxxxx[Y] Yusim2004 Cw*0707 x[RHK]xxxxxx[YL] Yusim2004 Cw*0708x[RQ]xxxxxx[YL] Yusim2004 Cw*0709 x[RHK]xxxxxx[YL] Yusim2004 Cw*0710x[YP]xxxxxx[FWY] Yusim2004 Cw*0711 x[R]xxxxxx[LM] Yusim2004 Cw*0712x[R]xxxxxx[LM] Yusim2004 Cw*0801 x[A]xxxxxx[LM] Yusim2004 Cw*0802x[A]xxxxxx[LM] Yusim2004 Cw*0803 x[A]xxxxxx[LM] Yusim2004 Cw*0804x[A]xxxxxx[LM] Yusim2004 Cw*0805 x[A]xxxxxx[LM] Yusim2004 Cw*0806x[A]xxxxxx[LM] Yusim2004 Cw*1202 x[A]xxxxxx[FWY] Yusim2004 Cw*1203x[A]xxxxxx[FWY] Yusim2004 Cw*1204 x[A]xxxxxx[L] Yusim2004 Cw*1205x[A]xxxxxx[L] Yusim2004 Cw*1206 x[A]xxxxxx[FWY] Yusim2004 Cw*1402x[YP]xxxxxx[FWY] Yusim2004 Cw*1403 x[YP]xxxxxx[FWY] Yusim2004 Cw*1404x[YP]xxxxxx[FWY] Yusim2004 Cw*1502 x[A]xxxxxx[LMYF] Yusim2004 Cw*1503x[A]xxxxxx[LMYF] Yusim2004 Cw*1504 x[A]xxxxxx[L] Yusim2004 Cw*1505x[A]xxxxxx[L] Yusim2004 Cw*1506 x[A]xxxxxx[LM] Yusim2004 Cw*1507x[A]xxxxxx[LMY] Yusim2004 Cw*1601 x[A]xxxxxx[FWY] Yusim2004 Cw*1602x[A]xxxxxx[L] Yusim2004 Cw*1604 x[A]xxxxxx[L] Yusim2004 Cw*1701x[A]xxxxxx[L] Yusim2004 Cw*1702 x[A]xxxxxx[L] Yusim2004 Cw*1801x[RQ]xxxxxx[LY] Yusim2004 Cw*1802 x[RQ]xxxxxx[LY] Yusim2004

The invention claimed is:
 1. A complex comprising an MHCI having apeptide derivative bound within its antigen-binding groove, wherein thepeptide derivative is selected from the group consisting of: (a) apeptide derivative having the general formula A-X_(aa)GIGILTV (SEQ IDNO:3), wherein X_(aa) is a non-natural amino acid that providesincreased conformational constraint in the peptide derivative comparedto the conformational constraint present in the parent peptide havingamino acid sequence AAGIGILTV (SEQ ID NO:1), wherein X_(aa) is selectedfrom the group consisting of [c3a], [c5g], [chg], [cpg], [dfb], [dhl],[phg], and [sta]; (b) a peptide derivative having the general formulaE-Xaa-AGIGILTV (SEQ ID NO:12), wherein Xaa is a non-natural amino acidthat provides increased conformational constraint in the peptidederivative compared to the conformational constraint present in theparent peptide having amino acid sequence EAAGIGILTV (SEQ ID NO:2),wherein Xaa is selected from the group consisting of [c3a], [c5g],[chg], [cpg], [dfb], [dhl], [phg], and [sta]; (c) a peptide derivativehaving the general formula I-X_(aa)-TFKNWPF (SEQ ID NO:22), whereinX_(aa) is a non-natural amino acid that provides increasedconformational constraint in the peptide derivative compared to theconformational constraint present in the parent peptide having aminoacid sequence ISTFKNWPF (SEQ ID NO:21), wherein X_(aa) is selected fromthe group consisting of [c3a], [c5g], [chg], [cpg], [dfb], [dhl], [phg],and [sta]; and (d) a peptide derivative having the general formulaK-X_(aa)-RRAIEQL (SEQ ID NO:32), wherein X_(aa) is a non-natural aminoacid that provides increased conformational constraint in the peptidederivative compared to the conformational constraint present in theparent peptide having amino acid sequence KVRRAIEQL (SEQ ID NO:31),wherein X_(aa) is selected from the group consisting of [c3a], [c5g],[chg], [cpg], [dfb], [dhl], [phg], and [sta].
 2. The complex of claim 1,wherein the peptide derivative is peptide derivative (a).
 3. The complexof claim 2, wherein the peptide derivative is A-[cpg]-GIGILTV (SEQ IDNO:7).
 4. The complex of claim 1, wherein the peptide derivative ispeptide derivative (b).
 5. The complex of claim 4, wherein the peptidederivative is selected from the group consisting of E-[chg]-AGIGILTV(SEQ ID NO:15); E-[cpg]-AGIGILTV (SEQ ID NO:16); and E-[dhl]-AGIGILTV(SEQ ID NO:18).
 6. The complex of claim 1, wherein the peptidederivative is peptide derivative (c).
 7. The complex of claim 6, whereinthe peptide derivative is I-[cpg]-TFKNWPF (SEQ ID NO:26).
 8. The complexof claim 1, wherein the peptide derivative is peptide derivative (d). 9.The complex of claim 8, wherein the peptide derivative isK-[cpg]-RRAIEQL (SEQ ID NO:36).